Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula.
It was reported recently that the plastid-located glutamine synthetase (GS2) from Medicago truncatula is regulated by phosphorylation catalysed by a calcium-dependent protein kinase and 14-3-3 interaction. Here it is shown that the two cytosolic GS isoenzymes, GS1a and GS1b, are also regulated by phosphorylation but, in contrast to GS2, GS1 phosphorylation is catalysed by calcium-independent kinase(s) and the phosphorylated enzymes fail to interact with 14-3-3s. Phosphorylation of GS1a occurs at more than one residue and was found to increase the affinity of the enzyme for the substrate glutamate. In vitro phosphorylation assays were used to compare the activity of GS kinase, present in different plant organs, against the three M. truncatula GS isoenzymes. All three GS proteins were phosphorylated by kinases present in leaves, roots, and nodules, but to different extents, suggesting a differential regulation under different metabolic contexts. Cytosolic GS phosphorylation was found to be affected by light in leaves and by active nitrogen fixation in root nodules, whereas GS2 phosphorylation was unaffected by these conditions. Some putative GS-binding phosphoproteins were identified showing both isoenzyme and organ specificity. Two phosphoproteins of 70 and 72 kDa were specifically bound to the cytosolic GS isoenzymes. Interestingly, phosphorylation of these proteins was also influenced by the nitrogen-fixing status of the nodule, suggesting that their phosphorylation and/or binding to GS are related to nitrogen fixation. Taken together, the results presented indicate that GS phosphorylation is modulated by nitrogen fixation in root nodules; these findings open up new possibilities to explore the involvement of this post-translational mechanism in nodule functioning.
Lima
Lígia
L
Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.
Seabra
Ana
A
Melo
Paula
P
Cullimore
Julie
J
Carvalho
Helena
H
D048948:14-3-3 Proteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D018506:Gene Expression Regulation, Plant; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008027:Light; D046913:Medicago truncatula / Q000201:enzymology* / Q000235:genetics; D009586:Nitrogen Fixation; D010766:Phosphorylation; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational
D048948:14-3-3 Proteins; D007527:Isoenzymes; D010940:Plant Proteins; D005974:Glutamate-Ammonia Ligase; D002118:Calcium