Novel aspects of glutamine synthetase (GS) regulation revealed by a detailed expression analysis of the entire GS gene family of Medicago truncatulaunder different physiological conditions

Abstract

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.

Transcriptome and metabolome of synthetic Solanum autotetraploids reveal key genomic stress events following polyploidization

“…Consistent with transcriptome data, most of the metabolite alterations were stochastic, rather than common. In S. commersonii tetraploids, glutamic acid, a key molecule in amino acid metabolism (Forde & Lea, 2007), was increased, while leucine and glutamine, which play essential roles in plant development, stress responses and nitrogen assimilation (Mentzen et al, 2008;Seabra et al, 2013;Yu et al, 2013), were reduced. Several transcripts were correlated with the changes in glutamate and glutamine concentrations, such as alanine/aspartate branched-chain amino acid transaminases (Liepman & Olsen, 2003).…”

Impact of concurrent overexpression of cytosolic glutamine synthetase (GS1) and sucrose phosphate synthase (SPS) on growth and development in transgenic tobacco

“…The inconsistency that has been observed in the literature with regards to the outcome of overexpressing GS 1 has been attributed to the transgene, the recipient of the transgene, and the growth conditions, specifically the availability and nature of N nutrients (Eckes et al 1989;Temple et al 1993;Oliveira et al 2002;Ortega et al 2004;Kirby et al 2006). GS besides being regulated transcriptionally, is also subject to post-transcriptional (Ortega et al 2006) and post-translational regulation (Ortega et al 1999;Lima et al 2006;Seabra et al 2013), but how these regulatory steps cause prolonged life span, delayed senescence and late flowering, is not known.…”

Nitrogen modulation of Medicago truncatula resistance to Aphanomyces euteiches depends on plant genotype

“…This highlighted that the SA022322 and F83005.5 genotypes responded differently to N deficiency despite both displaying NØ-IS. We analysed the expression of genes involved in N metabolism: MtNIA1 and MtNIA2, which encode two isoforms of NR; MtNRT2.1 and MtNRT2.3, which encode nitrate transporters (Pellizzaro et al, 2015); MtGS1a, MtGS1b and MtGS2a, which encode the two cytosolic and major plastid-located Gln synthetase enzymes, respectively (Seabra et al, 2013).…”