Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds

Abstract

Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed crossimmunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues.Dear Sirs, Please find enclosed a copy of our revised manuscript entitled "Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds" by M.A.Troncoso-Ponce, J. Rivoal, M. Venegas-Calerón, S. Dorion, R. Sánchez, F.J. Cejudo, R. Garcés and E. Martínez-Force to be consider its publication in your journal. This work describes by the first time the cloning, biochemical characterization, expression studies and localisation of three phosphoglycerate kinases, two cytosolic and one plastidial isoforms, from sunflower (Helianthus annuus L. We are sending to you a revised version of our manuscript PHYTOCHEM-D-12-00046R1, entitled "Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds", with amendments according to your comments and those from the referees, by M.A. Troncoso-Ponce, J. Rivoal, M. Venegas-Calerón, S. Dorion, R. Sánchez, F.J. Cejudo, R. Garcés and E. Martínez-Force to be considered its publication in your journal. Firstly, I would like to thank you and the reviewers to help us improving our manuscript.Reviewer(s)' Comments to Author:Reviewer #1: This manuscript is a resubmission of a paper describing the cloning, expression and kinetic characterisation of three isozymes of phosphoglycerate kinase (PGK) from developing sunflower seeds. The revised version addresses essentially all of the points raised in the initial review of the manuscript and provides a much more clearly focussed report on what remains a robust and informative study of the characteristics of a single step in glycolysis in a commercially important crop species. The paper is likely to provide a useful addition to the rather limited knowledge of the enzymology of carbohydrate metabolism in heterotrophic plant tissues.Although the authors have done a good job of revising the manuscript...

Cloning of theArabidopsisrwm1gene for resistance toWatermelon mosaic viruspoints to a new function for natural virus resistance genes

“…PGK proteins are evolutionary and structurally conserved enzymes that play a key role in cell metabolism (Watson et al ., ; Davies et al ., ; Szilagyi et al ., ; Troncoso‐Ponce et al ., ). In plants, at least two PGK isoforms occur in two different cellular compartments, one in the cytosol and one in chloroplasts (Shah and Bradbeer, ).…”

Phosphoglycerate Kinases Are Co-Regulated to Adjust Metabolism and to Optimize Growth

“…However, this latter assumption has not been thoroughly investigated to date. Subsequently, a second cytosolic PGK isoform from sunflower (Helianthus annuus) was cloned (Troncoso-Ponce et al, 2012), and in the Arabidopsis (Arabidopsis thaliana) genome, an additional putative PGK isoform with an N-terminal plastid/chloroplast localization signal was identified (Ouibrahim et al, 2014). The presence of two PGKs in the plastid/chloroplast could lead to specialization, so that one of them could be involved in photosynthesis and the other in glycolysis, which seems to be the case of GAPDH isoforms (Anoman et al, 2015).…”

“…Specifically, PGK2 could mediate the transport of viruses to the chloroplast (Lin et al, 2007;Cheng et al, 2013). The in vitro regulation of some PGK isoforms has been studied (Troncoso-Ponce et al, 2012;Morisse et al, 2014). It has been shown that the chloroplastic isoform of Chlamydomonas reinhardtii could be light regulated by thioredoxins (Morisse et al, 2014).…”

The Coordination of Gene Expression within Photosynthesis Pathway for Acclimation of C4 Energy Crop Miscanthus lutarioriparius

“…Transcripts of C 4 enzymes used by Wang et al ( 2009 ) and Calvin enzymes used by Reyes-Prieto and Troncoso-Ponce in dark reaction as well as their isoforms in S. bicolor, Oryza sativa, Z. mays , and Arabidopsis thaliana (Reyes-Prieto and Bhattacharya, 2007 ; Troncoso-Ponce et al, 2012 ) were downloaded from NCBI or Phytozome 9.1. Besides, Transcripts of three species, O. sativa, Z. mays , and A. thaliana , in relative to Calvin enzyme families collected in PMN ( http://www.plantcyc.org/ ) were downloaded from Phytozome 9.1.…”