Nitric oxide (NO) is emerging as an important regulatory player in the Rhizobium-legume symbiosis, but its biological role in nodule functioning is still far from being understood. To unravel the signal transduction cascade and ultimately NO function, it is necessary to identify its molecular targets. This study provides evidence that glutamine synthetase (GS), a key enzyme for root nodule metabolism, is a molecular target of NO in root nodules of Medicago truncatula, being regulated by tyrosine (Tyr) nitration in relation to active nitrogen fixation. In vitro studies, using purified recombinant enzymes produced in Escherichia coli, demonstrated that the M. truncatula nodule GS isoenzyme (MtGS1a) is subjected to NO-mediated inactivation through Tyr nitration and identified Tyr-167 as the regulatory nitration site crucial for enzyme inactivation. Using a sandwich enzymelinked immunosorbent assay, it is shown that GS is nitrated in planta and that its nitration status changes in relation to active nitrogen fixation. In ineffective nodules and in nodules fed with nitrate, two conditions in which nitrogen fixation is impaired and GS activity is reduced, a significant increase in nodule GS nitration levels was observed. Furthermore, treatment of root nodules with the NO donor sodium nitroprusside resulted in increased in vivo GS nitration accompanied by a reduction in GS activity. Our results support a role of NO in the regulation of nitrogen metabolism in root nodules and places GS as an important player in the process. We propose that the NO-mediated GS posttranslational inactivation is related to metabolite channeling to boost the nodule antioxidant defenses in response to NO.

The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance.

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.

In this report we demonstrate that plastid glutamine synthetase of Medicago truncatula (MtGS2) is regulated by phosphorylation and 14-3-3 interaction. To investigate regulatory aspects of GS2 phosphorylation, we have produced non-phosphorylated GS2 proteins by expressing the plant cDNA in E. coli and performed in vitro phosphorylation assays. The recombinant isoenzyme was phosphorylated by calcium dependent kinase(s) present in leaves, roots and nodules. Using an (His)6-tagged 14-3-3 protein column affinity purification method, we demonstrate that phosphorylated GS2 interacts with 14-3-3 proteins and that this interaction leads to selective proteolysis of the plastid located isoform, resulting in inactivation of the isoenzyme. By site directed mutagenesis we were able to identify a GS2 phosphorylation site (Ser97) crucial for the interaction with 14-3-3s. Phosphorylation of this target residue can be functionally mimicked by replacing Ser97 by Asp, indicating that the introduction of a negative charge contributes to the interaction with 14-3-3 proteins and subsequent specific proteolysis. Furthermore, we document that plant extracts contain protease activity that cleaves the GS2 protein only when it is bound to 14-3-3 proteins following either phosphorylation or mimicking of phosphorylation by Ser97Asp.

Glutamine synthetase (GS) is a vital enzyme for the assimilation of ammonia into amino acids in higher plants. In legumes, GS plays a crucial role in the assimilation of the ammonium released by nitrogen-fixing bacteria in root nodules, constituting an important metabolic knob controlling the nitrogen (N) assimilatory pathways. To identify new regulators of nodule metabolism, we profiled the transcriptome of Medicago truncatula nodules impaired in N assimilation by specifically inhibiting GS activity using phosphinothricin (PPT). Global transcript expression of nodules collected before and after PPT addition (4, 8, and 24 h) was assessed using Affymetrix M. truncatula GeneChip arrays. Hundreds of genes were regulated at the three time points, illustrating the dramatic alterations in cell metabolism that are imposed on the nodules upon GS inhibition. The data indicate that GS inhibition triggers a fast plant defense response, induces premature nodule senescence, and promotes loss of root nodule identity. Consecutive metabolic changes were identified at the three time points analyzed. The results point to a fast repression of asparagine synthesis and of the glycolytic pathway and to the synthesis of glutamate via reactions alternative to the GS/GOGAT cycle. Several genes potentially involved in the molecular surveillance for internal organic N availability are identified and a number of transporters potentially important for nodule functioning are pinpointed. The data provided by this study contributes to the mapping of regulatory and metabolic networks involved in root nodule functioning and highlight candidate modulators for functional analysis.

BackgroundGlutamine Synthetase (GS, EC 6.3.1.2) is a central enzyme in nitrogen metabolism, and a key component of nitrogen use efficiency (NUE) and plant yield and thus it is extremely important to understand how it is regulated in plants. Medicago truncatula provides an excellent model system to study GS, as it contain a very simple GS gene family comprising only four expressed genes, MtGS1a and MtGS1b encoding cytosolic polypeptides, and MtGS2a and MtGS2b encoding plastid-located enzymes. To identify new regulatory mechanisms controlling GS activity, we performed a detailed expression analysis of the entire GS gene family of M. truncatula in the major organs of the plant, over a time course of nodule or seed development and during a diurnal cycle.ResultsIndividual GS transcripts were quantified by qRT-PCR, and GS polypeptides and holoenzymes were evaluated by western blot and in-gel activity under native electrophoresis. These studies revealed that all four GS genes are differentially regulated in each organ of the plant, in a developmental manner, and identified new regulatory controls, which appear to be specific to certain metabolic contexts. Studies of the protein profiles showed that the GS polypeptides assemble into organ-specific protein complexes and suffer organ-specific post-translational modifications under defined physiological conditions. Our studies also reveal that GS expression and activity are modulated during a diurnal cycle. The biochemical properties of the four isoenzymes were determined and are discussed in relation to their function in the plant.ConclusionsThis work provides a comprehensive overview of GS expression and regulation in the model legume M. truncatula, contributing to a better understanding of the specific function of individual isoenzymes and to the identification of novel organ-specific post-translational mechanisms of GS regulation. We demonstrate that the GS proteins are modified and/or integrated into protein-complexes that assemble into a specific composition in particular organs of the plant. Taken together, the results presented here open new avenues to explore the regulatory mechanisms controlling GS activity in plants, a subject of major importance due to the crucial importance of the enzyme for plant growth and productivity.

Background Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula . Results This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2) in M. truncatula . The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene. Conclusions This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate that this gene duplication represents a functional innovation of plastid located GS related to storage protein accumulation exclusive to legume seed metabolism.

The condensation of ammonium and glutamate into glutamine catalyzed by glutamine synthetase (GS) is a fundamental step in nitrogen metabolism in all kingdoms of life. In plants, this is preceded by the reduction of inorganic nitrogen to an ammonium ion and therefore effectively articulates nitrogen fixation and metabolism. Although the three-dimensional structure of the dodecameric bacterial GS was determined quite some time ago, the quaternary architecture of the plant enzyme has long been assumed to be octameric, mostly on the basis of low-resolution electron-microscopy studies. Recently, the crystallographic structure of a monocotyledonous plant GS was reported that revealed a homodecameric organization. In order to unambiguously establish the quaternary architecture of GS from dicotyledonous plants, GS1a from the model legume Medicago truncatula was overexpressed, purified and crystallized. The collection of synchrotron diffraction data to 2.35 Å resolution allowed the determination of the three-dimensional structure of this enzyme by molecular replacement.