title: Over-expression of Arabidopsis thaliana carotenoid hydroxylases individually and in combination with a beta-carotene ketolase provides insight into in vivo functions.
abstract: Carotenoids represent a group of widely distributed pigments derived from the general isoprenoid biosynthetic pathway that possess diverse functions in plant primary and secondary metabolism. Modification of alpha- and beta-carotene backbones depends in part on ring hydroxylation. Two ferredoxin-dependent non-heme di-iron monooxygenases (AtB1 and AtB2) that mainly catalyze in vivo beta-carotene hydroxylations of beta,beta-carotenoids, and two heme-containing cytochrome P450 (CYP) monooxygenases (CYP97A3 and CYP97C1) that preferentially hydroxylate the epsilon-ring of alpha-carotene or the beta-ring of beta,epsilon-carotenoids, have been characterized in Arabidopsis by analysis of loss-of-function mutant phenotypes. We further investigated functional roles of both hydroxylase classes in modification of the beta- and epsilon-rings of alpha-carotene and beta-carotene through over-expression of AtB1, CYP97A3, CYP97C1, and the hydroxylase candidate CYP97B3. Since carotenoid hydroxylation is required for generation of ketocarotenoids by the bkt1(CrtO) beta-carotene ketolase, all hydroxylase constructs were also introduced into an Arabidopsis line expressing the Haematococcus pluvalis bkt1 beta-carotene ketolase. Analysis of foliar carotenoid profiles in lines overexpressing the individual hydroxylases indicate a role for CYP97B3 in carotenoid biosynthesis, confirm and extend previous findings of hydroxylase activities based on knock-out mutants, and suggest functions of the multifunctional enzymes in carotenoid biosynthesis. Hydroxylase over-expression in combination with bkt1 did not result in ketocarotenoid accumulation, but instead unexpected patterns of alpha-carotene derivatives, accompanied by a reduction of alpha-carotene, were observed. These data suggest possible interactions between the beta-carotene ketolase bkt1 and the hydroxylases that impact partitioning of carbon flux into different carotenoid branch pathways.
authors: Department of Animal Science, University of British Columbia, Vancouver, BC, Canada V6T1Z4.
authors: Ji-Eun
authors: JE
authors: Kim
authors: Kimberly M
authors: KM
authors: Cheng
authors: Neal E
authors: NE
authors: Craft
authors: Björn
authors: B
authors: Hamberger
authors: Carl J
authors: CJ
authors: Douglas
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002338:Carotenoids / Q000378:metabolism*; D015870:Gene Expression; D017343:Genes, Plant; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D019207:beta Carotene / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D019207:beta Carotene; D002338:Carotenoids; C041635:alpha-carotene; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C093259:beta-carotene ketolase; C079232:beta-carotene hydroxylase