title: Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.
abstract: The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants.
authors.affiliation: Instituto de la Grasa, CSIC, Edificio 46, Campus universitario Pablo de Olavide, Carretera de Utrera Km1, 41013 Sevilla, Spain.
authors.forename: Miriam
authors.initials: M
authors.lastname: Payá-Milans
authors.affiliation: Instituto de la Grasa, CSIC, Edificio 46, Campus universitario Pablo de Olavide, Carretera de Utrera Km1, 41013 Sevilla, Spain. Electronic address: mvc@ig.csic.es.
authors.forename: Mónica
authors.initials: M
authors.lastname: Venegas-Calerón
authors.affiliation: Instituto de la Grasa, CSIC, Edificio 46, Campus universitario Pablo de Olavide, Carretera de Utrera Km1, 41013 Sevilla, Spain.
authors.forename: Joaquín J
authors.initials: JJ
authors.lastname: Salas
authors.affiliation: Instituto de la Grasa, CSIC, Edificio 46, Campus universitario Pablo de Olavide, Carretera de Utrera Km1, 41013 Sevilla, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Garcés
authors.affiliation: Instituto de la Grasa, CSIC, Edificio 46, Campus universitario Pablo de Olavide, Carretera de Utrera Km1, 41013 Sevilla, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Martínez-Force
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D005994:Glycerophosphates; D006368:Helianthus / Q000201:enzymology* / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000737:chemistry; D018087:Plastids / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D005994:Glycerophosphates; C029620:alpha-glycerophosphoric acid; D005992:Glycerol-3-Phosphate O-Acyltransferase; C513677:plastidic glycerol-3-phosphate acyltransferase, Arabidopsis
keywords: Glycerol-3-phosphate acyltransferase; Helianthus annuus; Plastids; Substrate specificity; Sunflower