ORIGINAL ARTICLE Cloning, biochemical characterisation, tissue localisation and possible post-translational regulatory mechanism of the cytosolic phosphoglucose isomerase from developing sunflower seeds M. A. Troncoso-Ponce • J. Rivoal • F. J. Cejudo • S. Dorion • R. Garce ´s • E. Martı ´nez-Force Received: 27 April 2010 / Accepted: 23 June 2010 / Published online: 14 July 2010 Ó Springer-Verlag 2010 Abstract Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. One of the main sources of cellular NADPH is the oxidative pentose phosphate pathway (OPPP), generated from the oxidation of glucose-6-phosphate. This glycolytic inter- mediate, which can be imported to the plastid and enter in the OPPP, is the substrate and product of cytosolic phos- phoglucose isomerase (cPGI, EC 5.3.1.9). In this report, we describe the cloning of a full-length cDNA encoding cPGI from developing sunflower seeds. The sequence was pre- dicted to code for a protein of 566 residues characterised by the presence of two sugar isomerase domains. This cDNA was heterologously expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified using immobilised metal ion affinity chromatography and biochemically characterised. The enzyme had a spe- cific activity of 1,436 lmol min -1 mg -1 and 1,011 lmol min -1 mg -1 protein when the reaction was initiated with glucose-6-phosphate and fructose-6-phosphate, respec- tively. Activity was not affected by erythrose-4-phosphate, but was inhibited by 6-P gluconate and glyceraldehyde-3- phosphate. A polyclonal immune serum was raised against the purified enzyme, allowing the study of protein levels during the period of active lipid synthesis in seeds. These results were compared with PGI activity profiles and mRNA expression levels obtained from Q-PCR studies. Our results point to the existence of a possible post-trans- lational regulatory mechanism during seed development. Immunolocalisation of the protein in seed tissues further indicated that cPGI is highly expressed in the procambial ring. Keywords Cytosolic phosphoglucose isomerase Á Fructose-6-phosphate Á Gluconate Á Glucose-6-phosphate metabolism Á Glyceraldehyde-3-phosphate Á Sunflower Abbreviations F6P Fructose-6-phosphate G6P Glucose-6-phosphate GAP Glyceraldehyde-3-phosphate IPTG Isopropyl b-D-1-thiogalactopyranoside LB Luria–Bertani Ni-NTA Nickel-nitrilotriacetic acid PGI Phosphoglucose isomerase SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis TAG Triacylglycerides Introduction During the synthesis of reserve lipids in heterotrophic seeds, sucrose produced in the mother plant is metabolised by glycolytic enzymes (Schwender and Ohlrogge 2002; M. A. Troncoso-Ponce Á R. Garce ´s Á E. Martı ´nez-Force (&) Instituto de la Grasa, CSIC, Avda Padre Garcia Tejero 4, 41012 Seville, Spain e-mail: emforce@ig.csic.es J. Rivoal Á S. Dorion Institut de Recherche en Biologie Ve ´ge ´tale, Universite ´ de Montre ´al, 4101 Rue Sherbrooke est, Montre ´al, QC H1X 2B2, Canada F. J. Cejudo Instituto de Bioquı ´mica Vegetal y Fotosı ´ntesis, Universidad de Sevilla y CSIC, Avda Ame ´rico Vespucio 49, 41092 Seville, Spain 123 Planta (2010) 232:845–859 DOI 10.1007/s00425-010-1219-5