title: Functional analysis of the Brassica napus L. phytoene synthase (PSY) gene family.
abstract: Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three "Arabidopsis-like" subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed.
authors.affiliation: Genomics and Bioinformatics Unit, Agriaquaculture Nutritional Genomic Center, CGNA, Temuco, La Araucanía, Chile; Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Talca, Maule, Chile.
authors.forename: Ada
authors.initials: A
authors.lastname: López-Emparán
authors.affiliation: Genomics and Bioinformatics Unit, Agriaquaculture Nutritional Genomic Center, CGNA, Temuco, La Araucanía, Chile.
authors.forename: Daniela
authors.initials: D
authors.lastname: Quezada-Martinez
authors.affiliation: Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Talca, Maule, Chile.
authors.forename: Matías
authors.initials: M
authors.lastname: Zúñiga-Bustos
authors.affiliation: Depto. Cs. Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Región Metropolitana, Chile.
authors.forename: Víctor
authors.initials: V
authors.lastname: Cifuentes
authors.affiliation: Genomics and Bioinformatics Unit, Agriaquaculture Nutritional Genomic Center, CGNA, Temuco, La Araucanía, Chile.
authors.forename: Federico
authors.initials: F
authors.lastname: Iñiguez-Luy
authors.affiliation: Genomics and Bioinformatics Unit, Agriaquaculture Nutritional Genomic Center, CGNA, Temuco, La Araucanía, Chile.
authors.forename: María Laura
authors.initials: ML
authors.lastname: Federico
mesh_terms: D000595:Amino Acid Sequence; D029688:Brassica napus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002338:Carotenoids / Q000378:metabolism*; D017343:Genes, Plant; D008025:Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment
chemical_list: D010940:Plant Proteins; D002338:Carotenoids; C100185:(all-E) phytoene; D008025:Ligases