Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

Abstract

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.

Fertile transgenic Lotus corniculatus resistant to the non‐selective herbicide phosphinothricin

“…In L. japonicus , a closely related model legume, single GS2 and GS1 isoforms were detected with an in‐gel activity assay, as well as one hmGS protein in Western blots of native PAGE gels, which represented a dissociated GS2 tetramer with no activity (Betti et al , ). Generally, a small gene family (from two to four functional GLN1 genes) encoding for different cytosolic (GS1) isoforms and a single gene encoding for GS2 ( GLN2 ) are mostly present in plants (Betti et al , ), although it was recently shown that Medicago truncatula contains two functional GS2 genes (Seabra et al , ). Cytosolic isoforms are expressed in different plant organs, with specific roles in primary N assimilation in the root, N remobilization during senescence, N translocation in the vasculature (reviewed by Bernard & Habash, ).…”

Glutamine synthetase and glutamate dehydrogenase in triticale seeds: molecular cloning and genes expression

“…These isoforms include a cytosolic form, GS1, and a chloroplastic form, GS2 (Bielawski 1993;Miflin and Habash 2002). In general, there is only one gene encoding GS2, with the exception of Medicago truncatula for which two genes were cloned (Seabra et al 2010); however, a small, multi-gene family encodes for the GS1 proteins (Masclaux-Daubresse et al 2010). At this time, at least three GS1 genes in rice (Tabuchi et al 2005), five genes in maize (Martin et al 2006) and seven genes in wheat (Bernard et al 2008) have been identified.…”

Molecular and Cellular Analysis of Disease Resistance Mechanisms in Wheat

“…Glutamine synthetase (GS) is involved in the reassimilation of ammonium released by many processes such as deamination of amino acids, protein catabolism, and photorespiration (Seabra et al, 2010). During plant growth and development, nitrogen can be assimilated with the action of glutamine synthetase, forming glutamine.…”

“…Glutamine synthetase has two isoforms: a cytosolic GS1, which is involved in nitrogen remobilization, and a chloroplastic GS2, which is involved in nitrogen assimilation (Pegeau et al, 2006). GS is involved in the reassimilation of ammonium released by a number of biochemical processes such as protein catabolism, deamination of amino acids, and photorespiration (Seabra et al, 2010). The effects of SA and FB1 on glutamine synthetase activity have been studied in Roblin and Frontana.…”

“…In addition, during plant growth and development, nitrogen can be assimilated with the action of glutamine synthetase, forming glutamine. Transgenic approach has been applied in the attempt to enhance GS activity and to improve nitrogen assimilation(Man et al, 2011;Seabra et al, 2010). Ectopic expression of bacterial glutamine dehydrogenate in transgenic tobacco and corn was the main focus in order to enhance nitrogen utilization(Mungur et al, 2005), resulting in the ammonium incorporation into glutamine(Man et al, 2011).…”