title: An in vitro system for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation.
abstract: We have developed an assay based on rice embryogenic callus for rapid functional characterization of metabolic genes. We validated the assay using a selection of well-characterized genes with known functions in the carotenoid biosynthesis pathway, allowing rapid visual screening of callus phenotypes based on tissue color. We then used the system to identify the functions of two uncharacterized genes: a chemically synthesized β-carotene ketolase gene optimized for maize codon usage, and a wild-type Arabidopsis thaliana ortholog of the cauliflower Orange gene. In contrast to previous reports (Lopez, A.B., Van Eck, J., Conlin, B.J., Paolillo, D.J., O'Neill, J. and Li, L. () J. Exp. Bot. 59, 213-223; Lu, S., Van Eck, J., Zhou, X., Lopez, A.B., O'Halloran, D.M., Cosman, K.M., Conlin, B.J., Paolillo, D.J., Garvin, D.F., Vrebalov, J., Kochian, L.V., Küpper, H., Earle, E.D., Cao, J. and Li, L. () Plant Cell 18, 3594-3605), we found that the wild-type Orange allele was sufficient to induce chromoplast differentiation. We also found that chromoplast differentiation was induced by increasing the availability of precursors and thus driving flux through the pathway, even in the absence of Orange. Remarkably, we found that diverse endosperm-specific promoters were highly active in rice callus despite their restricted activity in mature plants. Our callus system provides a unique opportunity to predict the effect of metabolic engineering in complex pathways, and provides a starting point for quantitative modeling and the rational design of engineering strategies using synthetic biology. We discuss the impact of our data on analysis and engineering of the carotenoid biosynthesis pathway.
authors.affiliation: Department of Plant Production and Forestry Science, School of Agrifood and Forestry Science and Engineering (ETSEA), University of Lleida Agrotecnio Center, Avenida Alcalde Rovira Roure 191, 25198, Lleida, Spain.
authors.forename: Chao
authors.initials: C
authors.lastname: Bai
authors.forename: Sol M
authors.initials: SM
authors.lastname: Rivera
authors.forename: Vicente
authors.initials: V
authors.lastname: Medina
authors.forename: Rui
authors.initials: R
authors.lastname: Alves
authors.forename: Ester
authors.initials: E
authors.lastname: Vilaprinyo
authors.forename: Albert
authors.initials: A
authors.lastname: Sorribas
authors.forename: Ramon
authors.initials: R
authors.lastname: Canela
authors.forename: Teresa
authors.initials: T
authors.lastname: Capell
authors.forename: Gerhard
authors.initials: G
authors.lastname: Sandmann
authors.forename: Paul
authors.initials: P
authors.lastname: Christou
authors.forename: Changfu
authors.initials: C
authors.lastname: Zhu
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D053898:Biosynthetic Pathways; D002338:Carotenoids / Q000032:analysis / Q000378:metabolism*; D002454:Cell Differentiation; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant; D005838:Genotype; D060847:Metabolic Engineering; D055442:Metabolome; D055432:Metabolomics; D008962:Models, Theoretical; D012275:Oryza / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D010105:Oxygenases / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010940:Plant Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D018087:Plastids / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D014166:Transferases / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
chemical_list: D010940:Plant Proteins; D002338:Carotenoids; D010105:Oxygenases; C093259:beta-carotene ketolase; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase
keywords: 1-deoxy-d-xylulose 5-phosphate synthase; carotenoids; gene function; rice (Oryza sativa); technical advance; β-carotene ketolase