{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T01:15:01Z","timestamp":1780535701217,"version":"3.54.1"},"reference-count":126,"publisher":"Public Library of Science (PLoS)","issue":"1","license":[{"start":{"date-parts":[[2022,1,7]],"date-time":"2022-01-07T00:00:00Z","timestamp":1641513600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Cassava (<jats:italic>Manihot esculenta<\/jats:italic> Crantz) biofortification with provitamin A carotenoids is an ongoing process that aims to alleviate vitamin A deficiency. The moderate content of provitamin A carotenoids achieved so far limits the contribution to providing adequate dietary vitamin A levels. Strategies to increase carotenoid content focused on genes from the carotenoids biosynthesis pathway. In recent years, special emphasis was given to ORANGE protein (OR), which promotes the accumulation of carotenoids and their stability in several plants. The aim of this work was to identify, characterize and investigate the role of OR in the biosynthesis and stabilization of carotenoids in cassava and its relationship with phytoene synthase (PSY), the rate-limiting enzyme of the carotenoids biosynthesis pathway. Gene and protein characterization of OR, expression levels, protein amounts and carotenoids levels were evaluated in roots of one white (60444) and two yellow cassava cultivars (GM5309-57 and GM3736-37). Four OR variants were found in yellow cassava roots. Although comparable expression was found for three variants, significantly higher OR protein amounts were observed in the yellow varieties. In contrast, cassava <jats:italic>PSY1<\/jats:italic> expression was significantly higher in the yellow cultivars, but PSY protein amount did not vary. Furthermore, we evaluated whether expression of one of the variants, <jats:italic>MeOR_X1<\/jats:italic>, affected carotenoid accumulation in cassava Friable Embryogenic Callus (FEC). Overexpression of maize <jats:italic>PSY1<\/jats:italic> alone resulted in carotenoids accumulation and induced crystal formation. Co-expression with <jats:italic>MeOR_X1<\/jats:italic> led to greatly increase of carotenoids although <jats:italic>PSY1<\/jats:italic> expression was high in the co-expressed FEC. Our data suggest that posttranslational mechanisms controlling OR and PSY protein stability contribute to higher carotenoid levels in yellow cassava. Moreover, we showed that cassava FEC can be used to study the efficiency of single and combinatorial gene expression in increasing the carotenoid content prior to its application for the generation of biofortified cassava with enhanced carotenoids levels.<\/jats:p>","DOI":"10.1371\/journal.pone.0262412","type":"journal-article","created":{"date-parts":[[2022,1,7]],"date-time":"2022-01-07T18:30:57Z","timestamp":1641580257000},"page":"e0262412","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":17,"title":["Characterization of cassava ORANGE proteins and their capability to increase provitamin A carotenoids accumulation"],"prefix":"10.1371","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7463-6816","authenticated-orcid":true,"given":"Ang\u00e9lica M.","family":"Jaramillo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3608-0597","authenticated-orcid":true,"given":"Santiago","family":"Sierra","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul","family":"Chavarriaga-Aguirre","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0950-0074","authenticated-orcid":true,"given":"Diana Katherine","family":"Castillo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anestis","family":"Gkanogiannis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luis Augusto Becerra","family":"L\u00f3pez-Lavalle","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan Pablo","family":"Arciniegas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tianhu","family":"Sun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ralf","family":"Welsch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erick","family":"Boy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5930-2295","authenticated-orcid":true,"given":"Daniel","family":"\u00c1lvarez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,1,7]]},"reference":[{"key":"pone.0262412.ref001","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1007\/978-3-319-43739-2_9","volume-title":"Nutrition and Health in a Developing World","author":"AC Palmer","year":"2017"},{"key":"pone.0262412.ref002","doi-asserted-by":"crossref","unstructured":"Dubock A. Golden rice: to combat vitamin A deficiency for public health. Vitamin A. 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Cell Press"},{"key":"pone.0262412.ref008","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.plipres.2018.04.004","article-title":"A global perspective on carotenoids: Metabolism, biotechnology, and benefits for nutrition and health","author":"M Rodriguez-Concepcion","year":"2018","journal-title":"Progress in Lipid Research. 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Food Biofortification\u2014Reaping the Benefits of Science to Overcome Hidden Hunger. Council for Agricultural Science and Technology Issue Paper. 2020."},{"key":"pone.0262412.ref021","doi-asserted-by":"crossref","first-page":"S31","DOI":"10.1177\/15648265110321S105","article-title":"Biofortification: A new tool to reduce micronutrient malnutrition","volume":"32","author":"HE Bouis","year":"2011","journal-title":"Food and Nutrition Bulletin"},{"key":"pone.0262412.ref022","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1016\/j.worlddev.2009.03.014","article-title":"How cost-effective is biofortification in combating micronutrient malnutrition? An ex ante assessment","volume":"38","author":"J V. Meenakshi","year":"2010","journal-title":"World Development"},{"key":"pone.0262412.ref023","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.gfs.2017.01.009","article-title":"Improving nutrition through biofortification: A review of evidence from HarvestPlus, 2003 through 2016","volume":"12","author":"HE Bouis","year":"2017","journal-title":"Global food security"},{"key":"pone.0262412.ref024","doi-asserted-by":"crossref","unstructured":"Ceballos H, Davrieux F, Talsma EF, Belalcazar J, Chavarriaga P, Andersson MS. Carotenoids in cassava roots. Carotenoids. InTech; 2017.","DOI":"10.5772\/intechopen.68279"},{"key":"pone.0262412.ref025","doi-asserted-by":"crossref","first-page":"1251","DOI":"10.1007\/s00425-010-1250-6","article-title":"Characterization of phytoene synthases from cassava and their involvement in abiotic stress-mediated responses","volume":"232","author":"J Arango","year":"2010","journal-title":"Planta"},{"key":"pone.0262412.ref026","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.plantsci.2014.12.017","article-title":"Evolution of root-specific carotenoid precursor pathways for apocarotenoid signal biogenesis","volume":"233","author":"MH Walter","year":"2015","journal-title":"Plant Science"},{"key":"pone.0262412.ref027","doi-asserted-by":"crossref","first-page":"3348","DOI":"10.1105\/tpc.110.077560","article-title":"Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene","volume":"22","author":"R Welsch","year":"2010","journal-title":"The Plant Cell"},{"key":"pone.0262412.ref028","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1007\/s11248-017-0037-y","article-title":"Molecular analysis of the expression of a crtB transgene and the endogenous psy2-y 1 and psy2-y 2 genes of cassava and their effect on root carotenoid content","volume":"26","author":"P Chavarriaga-Aguirre","year":"2017","journal-title":"Transgenic Research"},{"key":"pone.0262412.ref029","doi-asserted-by":"crossref","first-page":"1186","DOI":"10.1111\/pbi.12862","article-title":"Provitamin A biofortification of cassava enhances shelf life but reduces dry matter content of storage roots due to altered carbon partitioning into starch","volume":"16","author":"G Beyene","year":"2018","journal-title":"Plant Biotechnology Journal"},{"key":"pone.0262412.ref030","first-page":"253","article-title":"Estudio preliminar de herencia del contenido de carotenoides en ra\u00edces de poblaciones segregantes de yuca 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plants","volume":"65","author":"P Schaub","year":"2017","journal-title":"Journal of Agricultural and Food Chemistry"},{"key":"pone.0262412.ref034","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1016\/j.foodchem.2017.03.158","article-title":"Carotenoid retention in biofortified maize using different post-harvest storage and packaging methods","volume":"232","author":"V Taleon","year":"2017","journal-title":"Food Chemistry"},{"key":"pone.0262412.ref035","doi-asserted-by":"crossref","first-page":"kiaa033","DOI":"10.1093\/plphys\/kiaa033","article-title":"Plant apocarotenoid metabolism utilizes defense mechanisms against reactive carbonyl species and xenobiotics","author":"J Koschmieder","year":"2020","journal-title":"Plant Physiology"},{"key":"pone.0262412.ref036","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1016\/j.jfca.2015.06.002","article-title":"Carotenoid stability during storage of yellow gari made from biofortified cassava or with palm oil","volume":"44","author":"A Bechoff","year":"2015","journal-title":"Journal of Food Composition and Analysis"},{"key":"pone.0262412.ref037","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1002\/jsfa.2704","article-title":"Retention of carotenoids in cassava roots submitted to different processing methods","volume":"87","author":"AL Ch\u00e1vez","year":"2007","journal-title":"Journal of the Science of Food and Agriculture"},{"key":"pone.0262412.ref038","doi-asserted-by":"crossref","first-page":"1344","DOI":"10.1021\/jf803053d","article-title":"Impact of style of processing on retention and bioaccessibility of \u03b2-carotene in cassava (Manihot esculenta, Crantz)","volume":"57","author":"SK Thakkar","year":"2009","journal-title":"Journal of Agricultural and Food Chemistry"},{"key":"pone.0262412.ref039","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1016\/j.foodres.2011.10.001","article-title":"Spatial distribution of dry matter in yellow fleshed cassava roots and its influence on carotenoid retention upon boiling","volume":"45","author":"H Ceballos","year":"2012","journal-title":"Food Research International"},{"key":"pone.0262412.ref040","doi-asserted-by":"crossref","first-page":"1434","DOI":"10.1002\/jsfa.9347","article-title":"Carotenoids retention in biofortified yellow cassava processed with traditional African methods","volume":"99","author":"V Taleon","year":"2019","journal-title":"Journal of the Science of Food and Agriculture"},{"key":"pone.0262412.ref041","first-page":"82","volume-title":"Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc","author":"Institute of Medicine Food and Nutrition Board","year":"2001"},{"key":"pone.0262412.ref042","doi-asserted-by":"crossref","first-page":"1344","DOI":"10.3389\/fpls.2016.01344","article-title":"Differential contribution of the first two enzymes of the MEP pathway to the supply of metabolic precursors for carotenoid and chlorophyll biosynthesis in carrot (Daucus carota)","volume":"7","author":"K Simpson","year":"2016","journal-title":"Frontiers in Plant Science"},{"key":"pone.0262412.ref043","doi-asserted-by":"crossref","first-page":"737","DOI":"10.1007\/s00425-016-2637-9","article-title":"Carotenoid gene expression explains the difference of carotenoid accumulation in carrot root tissues","volume":"245","author":"F Perrin","year":"2017","journal-title":"Planta"},{"key":"pone.0262412.ref044","doi-asserted-by":"crossref","DOI":"10.3390\/ijms18122515","article-title":"The silencing of carotenoid \u03b2-hydroxylases by RNA interference in different maize genetic backgrounds increases the \u03b2 carotene content of the endosperm","volume":"18","author":"J Berman","year":"2017","journal-title":"International Journal of Molecular 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Sun","year":"2018","journal-title":"Molecular Plant"},{"key":"pone.0262412.ref048","article-title":"Carotenoid pigment accumulation in horticultural plants","author":"AS Hermanns","year":"2020","journal-title":"Horticultural Plant Journal"},{"key":"pone.0262412.ref049","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1007\/BF00147182","article-title":"A mutation affecting curd colour in cauliflower (Brassica oleracea L. var. Botrytis DC)","volume":"24","author":"P Crisp","year":"1975","journal-title":"Euphytica"},{"key":"pone.0262412.ref050","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1046\/j.1365-313x.2001.01008.x","article-title":"A novel gene mutation that confers abnormal patterns of \u03b2-carotene accumulation in cauliflower (Brassica oleracea var. botrytis)","volume":"26","author":"L Li","year":"2001","journal-title":"The Plant Journal"},{"key":"pone.0262412.ref051","doi-asserted-by":"crossref","first-page":"3594","DOI":"10.1105\/tpc.106.046417","article-title":"The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of \u03b2-carotene accumulation","volume":"18","author":"S Lu","year":"2006","journal-title":"the Plant Cell Online"},{"key":"pone.0262412.ref052","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3389\/fpls.2019.01716","article-title":"Characterization of cauliflower OR mutant variants","volume":"10","author":"R Welsch","year":"2020","journal-title":"Frontiers in Plant Science"},{"key":"pone.0262412.ref053","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1111\/tpj.12814","article-title":"A \u2018golden\u2019 SNP in CmOr governs the fruit flesh color of melon (Cucumis melo)","volume":"82","author":"G Tzuri","year":"2015","journal-title":"The Plant Journal"},{"key":"pone.0262412.ref054","article-title":"Carotenoid presence is associated with the or gene in domesticated carrot","author":"SL Ellison","year":"2018","journal-title":"Genetics"},{"key":"pone.0262412.ref055","doi-asserted-by":"crossref","first-page":"1599","DOI":"10.1007\/s00709-015-0919-x","article-title":"The DnaJ-like zinc finger domain protein ORANGE localizes to the nucleus in etiolated cotyledons of Arabidopsis thaliana","volume":"253","author":"TH Sun","year":"2016","journal-title":"Protoplasma"},{"key":"pone.0262412.ref056","doi-asserted-by":"crossref","DOI":"10.1073\/pnas.1420831112","article-title":"Arabidopsis OR 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Or gene enhances carotenoid accumulation and stability during post-harvest storage of potato tubers","volume":"5","author":"L Li","year":"2012","journal-title":"Molecular Plant"},{"key":"pone.0262412.ref060","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1093\/jxb\/erm299","article-title":"Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers","volume":"59","author":"AB Lopez","year":"2008","journal-title":"Journal of Experimental Botany"},{"key":"pone.0262412.ref061","doi-asserted-by":"crossref","DOI":"10.1038\/hortres.2015.36","article-title":"Carotenoid metabolism and regulation in horticultural crops","volume":"2","author":"H Yuan","year":"2015","journal-title":"Horticulture Research"},{"key":"pone.0262412.ref062","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1104\/pp.16.01256","article-title":"Distinct Mechanisms of the ORANGE Protein in Controlling Carotenoid Flux","volume":"173","author":"N Chayut","year":"2017","journal-title":"Plant Physiology"},{"key":"pone.0262412.ref063","doi-asserted-by":"crossref","first-page":"322","DOI":"10.1038\/ng.551","article-title":"Rare genetic variation at Zea mays crtRB1 increases \u03b2-carotene in maize grain","volume":"42","author":"J Yan","year":"2010","journal-title":"Nature Genetics"},{"key":"pone.0262412.ref064","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.phytochem.2011.11.003","article-title":"Down-regulation of \u03b2-carotene hydroxylase increases \u03b2-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato","volume":"74","author":"SH Kim","year":"2012","journal-title":"Phytochemistry"},{"key":"pone.0262412.ref065","first-page":"1","article-title":"Orange protein has a role in phytoene synthase stabilization in sweetpotato","volume":"6","author":"S Park","year":"2016","journal-title":"Scientific 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BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95\/98\/NT. Nucleic Acids Symposium Series. 1999. pp. 95\u201398."},{"key":"pone.0262412.ref085","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1007\/s12042-011-9088-z","article-title":"The Cassava Genome: Current Progress, Future Directions","volume":"5","author":"S Prochnik","year":"2012","journal-title":"Tropical Plant Biol"},{"key":"pone.0262412.ref086","doi-asserted-by":"crossref","first-page":"1754","DOI":"10.1093\/bioinformatics\/btp324","article-title":"Fast and accurate short read alignment with Burrows-Wheeler transform","volume":"25","author":"H Li","year":"2009","journal-title":"Bioinformatics"},{"key":"pone.0262412.ref087","doi-asserted-by":"crossref","first-page":"2156","DOI":"10.1093\/bioinformatics\/btr330","article-title":"The variant call format and VCFtools","volume":"27","author":"P Danecek","year":"2011","journal-title":"Bioinformatics"},{"key":"pone.0262412.ref088","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0006373","article-title":"Carotenoid crystal formation in Arabidopsis and carrot roots caused by increased phytoene synthase protein levels","volume":"4","author":"D Maass","year":"2009","journal-title":"PLoS ONE"},{"key":"pone.0262412.ref089","doi-asserted-by":"crossref","first-page":"10354","DOI":"10.1073\/pnas.0806042106","article-title":"Elimination of antiviral defense by viral RNase III","volume":"106","author":"WJ Cuellar","year":"2009","journal-title":"Proceedings of the National Academy of Sciences of the United States of America"},{"key":"pone.0262412.ref090","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1016\/j.ab.2009.06.004","article-title":"Multiple reprobing of Western blots after inactivation of peroxidase activity by its substrate, hydrogen peroxide","volume":"393","author":"AD Sennepin","year":"2009","journal-title":"Analytical Biochemistry"},{"key":"pone.0262412.ref091","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1038\/nmeth.2089","article-title":"NIH Image to ImageJ: 25 years of image analysis","volume":"9","author":"CA Schneider","year":"2012","journal-title":"Nature Methods"},{"key":"pone.0262412.ref092","doi-asserted-by":"crossref","first-page":"e0209702","DOI":"10.1371\/journal.pone.0209702","article-title":"A comparison study of five different methods to measure carotenoids in biofortified yellow cassava (Manihot esculenta)","volume":"13","author":"A Jaramillo","year":"2018","journal-title":"PLOS ONE"},{"key":"pone.0262412.ref093","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1104\/pp.108.117028","article-title":"A third phytoene synthase is devoted to abiotic stress-induced abscisic acid formation in rice and defines functional diversification of phytoene synthase genes","volume":"147","author":"R Welsch","year":"2008","journal-title":"Plant Physiology"},{"key":"pone.0262412.ref094","doi-asserted-by":"crossref","first-page":"673","DOI":"10.3389\/fpls.2019.00673","article-title":"Arabidopsis LEC1 and LEC2 Orthologous Genes Are Key Regulators of Somatic Embryogenesis in Cassava","volume":"10","author":"A Brand","year":"2019","journal-title":"Frontiers in Plant Science"},{"key":"pone.0262412.ref095","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1007\/s12042-012-9099-4","article-title":"A high-throughput platform for the production and analysis of transgenic cassava (Manihot esculenta) plants","volume":"5","author":"N Taylor","year":"2012","journal-title":"Tropical Plant Biology"},{"key":"pone.0262412.ref096","doi-asserted-by":"crossref","first-page":"482","DOI":"10.1038\/nbt1082","article-title":"Improving the nutritional value of Golden Rice through increased pro-vitamin A content","volume":"23","author":"JA Paine","year":"2005","journal-title":"Nature Biotechnology"},{"key":"pone.0262412.ref097","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1104\/pp.103.027979","article-title":"A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta","volume":"133","author":"MD Curtis","year":"2003","journal-title":"Plant Physiology"},{"key":"pone.0262412.ref098","first-page":"391","article-title":"Microbial \u03b2-glucuronidase genes, gene products and uses thereof","volume":"6","author":"R Jefferson","year":"2003","journal-title":"Google Patents. US Patent"},{"key":"pone.0262412.ref099","doi-asserted-by":"crossref","first-page":"1845","DOI":"10.1038\/nprot.2009.208","article-title":"Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava","volume":"4","author":"SE Bull","year":"2009","journal-title":"Nature Protocols"},{"key":"pone.0262412.ref100","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1007\/s002990050550","article-title":"Plant regeneration from protoplasts isolated from friable embryogenic callus of cassava","volume":"18","author":"E Sofiari","year":"1998","journal-title":"Plant Cell Reports"},{"key":"pone.0262412.ref101","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1007\/s42994-021-00046-1","article-title":"Multi-strategy engineering greatly enhances provitamin A carotenoid accumulation and stability in Arabidopsis seeds","volume":"2","author":"T Sun","year":"2021","journal-title":"aBIOTECH"},{"key":"pone.0262412.ref102","doi-asserted-by":"crossref","first-page":"992","DOI":"10.3945\/an.116.014720","article-title":"Vitamin A","volume":"8","author":"H Debelo","year":"2017","journal-title":"Advances in Nutrition: An International Review Journal"},{"key":"pone.0262412.ref103","doi-asserted-by":"crossref","first-page":"2551","DOI":"10.3945\/jn.116.239202","article-title":"Provitamin A carotenoid\u2013biofortified maize consumption increases pupillary responsiveness among zambian children in a randomized controlled trial","volume":"146","author":"AC Palmer","year":"2016","journal-title":"The Journal of Nutrition"},{"key":"pone.0262412.ref104","doi-asserted-by":"crossref","first-page":"258","DOI":"10.3945\/ajcn.114.100164","article-title":"Biofortified yellow cassava and vitamin A status of Kenyan children: a randomized controlled trial","volume":"103","author":"EF Talsma","year":"2016","journal-title":"American Journal of Clinical Nutrition"},{"key":"pone.0262412.ref105","doi-asserted-by":"crossref","first-page":"1871","DOI":"10.3945\/jn.111.151829","article-title":"Introduction of \u03b2-Carotene-rich orange sweet potato in rural Uganda resulted in increased vitamin a intakes among children and women and improved vitamin a status among children","volume":"142","author":"C Hotz","year":"2012","journal-title":"Journal of Nutrition"},{"key":"pone.0262412.ref106","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1017\/S0007114511005174","article-title":"A large-scale intervention to introduce orange sweet potato in rural Mozambique increases vitamin A intakes among children and women","volume":"108","author":"C Hotz","year":"2012","journal-title":"British Journal of Nutrition"},{"key":"pone.0262412.ref107","doi-asserted-by":"crossref","DOI":"10.3390\/foods8050177","article-title":"Retention of Pro-Vitamin A content in products from new biofortified cassava varieties","volume":"8","author":"TE Eyinla","year":"2019","journal-title":"Foods"},{"key":"pone.0262412.ref108","unstructured":"Ovalle T, Perea C, Pizarro M, Morante N, Ceballos H, Dufour D, et al. Elucidating high beta-carotene accumulation in cassava based on next generation sequencing. World Congress on Root and Tuber Crops. Nanning, Guangxi, China; 2016. pp. SP06-16."},{"key":"pone.0262412.ref109","doi-asserted-by":"crossref","first-page":"480","DOI":"10.1111\/nph.14827","article-title":"Novel DNAJ-related proteins in Arabidopsis thaliana","volume":"217","author":"P Pulido","year":"2017","journal-title":"New Phytologist"},{"key":"pone.0262412.ref110","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/j.gene.2019.03.071","article-title":"Genomics analysis of the light-harvesting chlorophyll a\/b-binding (Lhc) superfamily in cassava (Manihot esculenta Crantz)","volume":"702","author":"Z Zou","year":"2019","journal-title":"Gene"},{"key":"pone.0262412.ref111","doi-asserted-by":"crossref","first-page":"255","DOI":"10.3389\/fpls.2018.00255","article-title":"Strigolactone levels in dicot roots are determined by an ancestral symbiosis-regulated clade of the PHYTOENE SYNTHASE gene family","volume":"0","author":"R Stauder","year":"2018","journal-title":"Frontiers in Plant Science"},{"key":"pone.0262412.ref112","doi-asserted-by":"crossref","first-page":"3725","DOI":"10.1105\/tpc.112.104174","article-title":"Plastid localization of the key carotenoid enzyme phytoene synthase is altered by isozyme, allelic variation, and activity","volume":"24","author":"M Shumskaya","year":"2012","journal-title":"Plant Cell"},{"key":"pone.0262412.ref113","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1007\/s10681-015-1541-x","article-title":"Association of phytoene synthase Psy1-A1 and Psy1-B1 allelic variants with semolina yellowness in durum wheat (Triticum turgidum L. var. durum)","volume":"207","author":"KM Campos","year":"2016","journal-title":"Euphytica"},{"key":"pone.0262412.ref114","first-page":"1","article-title":"Genome sequences of two diploid wild relatives of cultivated sweetpotato reveal targets for genetic improvement","volume":"9","author":"S Wu","year":"2018","journal-title":"Nature Communications"},{"key":"pone.0262412.ref115","doi-asserted-by":"crossref","first-page":"1081","DOI":"10.1111\/pbi.13039","article-title":"Single-molecule real-time sequencing reveals diverse allelic variations in carotenoid biosynthetic genes in pepper (Capsicum spp.)","volume":"17","author":"HB Jeong","year":"2019","journal-title":"Plant Biotechnology Journal"},{"key":"pone.0262412.ref116","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1007\/s10681-016-1772-5","article-title":"Genome-wide association mapping of provitamin A carotenoid content in cassava","volume":"212","author":"W Esuma","year":"2016","journal-title":"Euphytica"},{"key":"pone.0262412.ref117","doi-asserted-by":"crossref","first-page":"0","DOI":"10.3835\/plantgenome2016.09.0094","article-title":"Genome-wide association mapping of correlated traits in cassava: dry matter and total carotenoid content","volume":"10","author":"IY Rabbi","year":"2017","journal-title":"Plant Genome"},{"key":"pone.0262412.ref118","article-title":"Candidate gene sequencing and validation of SNP markers linked to carotenoid content in cassava (Manihot esculenta Crantz)","volume":"37","author":"LI Udoh","year":"2017","journal-title":"Molecular Breeding"},{"key":"pone.0262412.ref119","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/j.molp.2017.11.003","article-title":"Clp Protease and OR Directly Control the Proteostasis of Phytoene Synthase, the Crucial Enzyme for Carotenoid Biosynthesis in Arabidopsis","volume":"11","author":"R Welsch","year":"2018","journal-title":"Molecular Plant"},{"key":"pone.0262412.ref120","doi-asserted-by":"crossref","first-page":"1988","DOI":"10.1104\/pp.19.00384","article-title":"A neighboring aromatic-aromatic amino acid combination governs activity divergence between tomato phytoene synthases","volume":"180","author":"H Cao","year":"2019","journal-title":"Plant Physiology"},{"key":"pone.0262412.ref121","first-page":"1","article-title":"Natural variation in expression of genes associated with carotenoid biosynthesis and accumulation in cassava (Manihot esculenta Crantz) storage root","volume":"16","author":"LJCB Carvalho","year":"2016","journal-title":"BMC Plant Biology"},{"key":"pone.0262412.ref122","doi-asserted-by":"crossref","unstructured":"Wahyuni Y, Anika M, Putri DH, Hartati NS, Harmoko R, Sudarmonowati E. Variation in transcriptional profiles of carotenoid biosynthetic genes in indonesian yellow- and white-fleshed tuberous root cassava (Manihot esculenta Crantz) accessions. IOP Conference Series: Earth and Environmental Science. 2020;439.","DOI":"10.1088\/1755-1315\/439\/1\/012016"},{"key":"pone.0262412.ref123","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1111\/j.1365-313X.2007.03362.x","article-title":"Abscisic acid deficiency in the tomato mutant high-pigment 3 leading to increased plastid number and higher fruit lycopene content","volume":"53","author":"N Galpaz","year":"2008","journal-title":"Plant Journal"},{"key":"pone.0262412.ref124","doi-asserted-by":"crossref","first-page":"3861","DOI":"10.1021\/jf204958w","article-title":"Retention during processing and bioaccessibility of \u03b2-carotene in high \u03b2-carotene transgenic cassava root","volume":"60","author":"ML Failla","year":"2012","journal-title":"Journal of Agricultural and Food Chemistry"},{"key":"pone.0262412.ref125","first-page":"1","article-title":"The organ-specific differential roles of rice DXS and DXR, the first two enzymes of the MEP pathway, in carotenoid metabolism in Oryza sativa leaves and seeds","volume":"20","author":"MK You","year":"2020","journal-title":"BMC Plant Biology"},{"key":"pone.0262412.ref126","doi-asserted-by":"crossref","first-page":"1455","DOI":"10.1007\/s00425-018-2988-5","article-title":"Unique chromoplast organisation and carotenoid gene expression in carotenoid-rich carrot callus","volume":"248","author":"T Oleszkiewicz","year":"2018","journal-title":"Planta"}],"updated-by":[{"DOI":"10.1371\/journal.pone.0263168","type":"correction","label":"Correction","source":"publisher","updated":{"date-parts":[[2022,1,21]],"date-time":"2022-01-21T00:00:00Z","timestamp":1642723200000}}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0262412","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,1,7]],"date-time":"2022-01-07T18:32:20Z","timestamp":1641580340000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0262412"}},"subtitle":[],"editor":[{"given":"Hiroshi","family":"Ezura","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2022,1,7]]},"references-count":126,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2022,1,7]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0262412","relation":{"correction":[{"id-type":"doi","id":"10.1371\/journal.pone.0263168","asserted-by":"object"}]},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,1,7]]}}}