Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula

Abstract

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.

Reduced light and moderate water deficiency sustain nitrogen assimilation and sucrose degradation at low temperature in durum wheat

“…amino acids. Both NR (Campbell, 1999) and GS reaction greatly depends on light which directly modulates the GS activity in leaves at transcriptional (Oliveira and Coruzzi, 1999) via photorepector-mediated way and posttranslational level via phosphorylation of the GS protein (Lima et al, 2006). The efficiency of photosynthesis is light-dependent and provide ATP and the carbon skeleton for amino acid synthesis.…”

14-3-3 Proteins SGF14c and SGF14l Play Critical Roles during Soybean Nodulation

“…Performing these activities, 14-3-3 proteins function as regulators of important biological processes in plants, such as development, metabolism, transcription, organellar protein trafficking, and stress responses (Bunney et al, 2001;Roberts et al, 2002;Sehnke et al, 2002;Roberts, 2003;Shi et al, 2007). In legume species such as Medicago truncatula, plastidlocated Gln synthetase (GS) is regulated by phosphorylation catalyzed by a calcium-dependent protein kinase and a 14-3-3 interaction when GS phosphorylation is mediated by nitrogen fixation in root nodules (Lima et al, 2006a(Lima et al, , 2006b.…”

Glutamine Synthetase Is a Molecular Target of Nitric Oxide in Root Nodules of Medicago truncatula and Is Regulated by Tyrosine Nitration

“…The M. truncatula GS isoenzymes MtGS1a and MtGS2a were produced in E. coli BL21-CodonPlus (DE3)-RP cells harboring previously described gene constructs pET28a-MtGS1a and pET28a-MtGS2a encoding N-terminally Histagged fusion proteins (Lima et al, 2006b).…”

“…The regulation of GS at the transcriptional level has been well studied, and it is generally accepted as the primary regulatory point, but a number of posttranslational regulatory controls were shown to be critical for the regulation of GS activity (Hoelzle et al, 1992;Temple et al, 1996Temple et al, , 1998Ortega et al, 1999Ortega et al, , 2001. In higher plants, GS activity is known to be modulated by oxidative turnover (Ortega et al, 1999;Palatnik et al, 1999;Ishida et al, 2002) and phosphorylation along with 14-3-3 interaction (Finnemann and Schjoerring, 2000;Man and Kaiser, 2001;Riedel et al, 2001;Lima et al, 2006aLima et al, , 2006b). In Escherichia coli, the regulation of GS activity involves cyclic adenylylation and nitration of Tyr residues (Berlet et al, 1996).…”