title: Knockout mutants as a tool to identify the subunit composition of Arabidopsis glutamine synthetase isoforms.
abstract: Glutamine synthetase (GS) is a key enzyme in nitrogen assimilation, which catalyzes the formation of glutamine from ammonia and glutamate. Plant GS isoforms are multimeric enzymes, recently shown to be decamers. The Arabidopsis genome encodes five cytosolic (GS1) proteins labeled as GLN1;1 through GLN1;5 and one chloroplastic (GS2) isoform, GLN2;0. However, as many as 11 GS activity bands were resolved from different Arabidopsis tissues by Native PAGE and activity staining. Western analysis showed that all 11 isoforms are composed exclusively of 40 kDa GS1 subunits. Of five GS1 genes, only GLN1;1, GLN1;2 and GLN1;3 transcripts accumulated to significant levels in vegetative tissues, indicating that only subunits encoded by these three genes produce the 11-band zymogram. Even though the GS2 gene also had significant expression, the corresponding activity was not detected, probably due to inactivation. To resolve the subunit composition of 11 active GS1 isoforms, homozygous knockout mutants deficient in the expression of different GS1 genes were selected from the progeny of T-DNA insertional SALK and SAIL lines. Comparison of GS isoenzyme patterns of the selected GS1 knockout mutants indicated that all of the detected isoforms consist of varying proportions of GLN1;1, GLN1;2 and GLN1;3 subunits, and that GLN1;1 and GLN1;3, as well as GLN1;2 and GLN1;3 and possibly GLN1;1 and GLN1;2 proteins combine in all proportions to form active homo- and heterodecamers.
authors.affiliation: Institute for Biological Research "Siniša Stanković", Department for Plant Physiology, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia. Electronic address: mdragicevic@ibiss.bg.ac.rs.
authors.forename: Milan
authors.initials: M
authors.lastname: Dragićević
authors.affiliation: Institute for Biological Research "Siniša Stanković", Department for Plant Physiology, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia. Electronic address: slatod@ibiss.bg.ac.rs.
authors.forename: Slađana
authors.initials: S
authors.lastname: Todorović
authors.affiliation: Institute for Biological Research "Siniša Stanković", Department for Plant Physiology, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia. Electronic address: milica84bog@gmail.com.
authors.forename: Milica
authors.initials: M
authors.lastname: Bogdanović
authors.affiliation: Institute for Biological Research "Siniša Stanković", Department for Plant Physiology, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia. Electronic address: biljana.nikolic@ibiss.bg.ac.rs.
authors.forename: Biljana
authors.initials: B
authors.lastname: Filipović
authors.affiliation: Institute for Biological Research "Siniša Stanković", Department for Plant Physiology, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia. Electronic address: dmisic@ibiss.bg.ac.rs.
authors.forename: Danijela
authors.initials: D
authors.lastname: Mišić
authors.affiliation: Institute for Biological Research "Siniša Stanković", Department for Plant Physiology, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia. Electronic address: ana.simonovic@ibiss.bg.ac.rs.
authors.forename: Ana
authors.initials: A
authors.lastname: Simonović
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000201:enzymology* / Q000235:genetics
chemical_list: D007527:Isoenzymes; D005974:Glutamate-Ammonia Ligase
keywords: Arabidopsis thaliana; Glutamine synthetase; Isoforms; Knockout mutants; Subunit composition; T-DNA insertion