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Cloning, biochemical characterisation, tissue localisation and possible post-translational regulatory mechanism of the cytosolic phosphoglucose isomerase from developing sunflower seeds

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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 intermediate, which can be imported to the plastid and enter in the OPPP, is the substrate and product of cytosolic phosphoglucose 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 predicted 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 specific activity of 1,436 μmol min−1 mg−1 and 1,011 μmol min−1 mg−1 protein when the reaction was initiated with glucose-6-phosphate and fructose-6-phosphate, respectively. 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-translational regulatory mechanism during seed development. Immunolocalisation of the protein in seed tissues further indicated that cPGI is highly expressed in the procambial ring.

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Abbreviations

F6P:

Fructose-6-phosphate

G6P:

Glucose-6-phosphate

GAP:

Glyceraldehyde-3-phosphate

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

LB:

Luria–Bertani

Ni-NTA:

Nickel-nitrilotriacetic acid

PGI:

Phosphoglucose isomerase

SDS-PAGE:

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

TAG:

Triacylglycerides

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Acknowledgments

Thanks are due to M. C. Ruiz for her skilful technical assistance. This work was supported by the Spanish Ministry of Science and Innovation and FEDER, project AGL2008-01086/ALI. It was also supported in part by a discovery Grant from the Natural Sciences and the Engineering Research Council of Canada to J. Rivoal. The work in the FJC laboratory was supported by Grant BIO2007-60644 from the Spanish Ministry of Education and Science, and Grants P06-CVI-01578 and BIO-182 from the Andalusian government (Spain).

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Authors and Affiliations

  1. Instituto de la Grasa, CSIC, Avda Padre Garcia Tejero 4, 41012, Seville, Spain

    M. A. Troncoso-Ponce, R. Garcés & E. Martínez-Force

  2. Institut de Recherche en Biologie Végétale, Université de Montréal, 4101 Rue Sherbrooke est, Montréal, QC, H1X 2B2, Canada

    J. Rivoal & S. Dorion

  3. Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla y CSIC, Avda Américo Vespucio 49, 41092, Seville, Spain

    F. J. Cejudo

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  1. M. A. Troncoso-Ponce
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  2. J. Rivoal
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  3. F. J. Cejudo
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  4. S. Dorion
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  5. R. Garcés
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  6. E. Martínez-Force
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Correspondence to E. Martínez-Force.

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Troncoso-Ponce, M.A., Rivoal, J., Cejudo, F.J. et al. Cloning, biochemical characterisation, tissue localisation and possible post-translational regulatory mechanism of the cytosolic phosphoglucose isomerase from developing sunflower seeds. Planta 232, 845–859 (2010). https://doi.org/10.1007/s00425-010-1219-5

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  • DOI: https://doi.org/10.1007/s00425-010-1219-5

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