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

BMC Plant Biology Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds Ana R Seabra 1 Cristina P Vieira 1 Julie V Cullimore 0 Helena G Carvalho 1 0 Laboratoire des Interactions Plantes-Microorganismes, Institut National de la Recherche Agronomique - Centre National de la Recherche Scientifique , Boite Postale 52627, 31326 Castanet-Tolosan Cedex , France 1 Instituto de Biologia Molecular e Celular da Universidade do Porto , Rua do Campo Alegre, 823, 4150-180 Porto , Portugal 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. - Background Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Nitrogen is assimilated in plants through the action of Glutamine Synthetase (GS, EC 6.3.1.2) forming glutamine, which serves as a building block for all nitrogen containing compounds in the plant. GS is a complex and highly regulated enzyme, which in addition to the primary ammonium assimilation, is involved in the * Correspondence: 1Instituto de Biologia Molecular e Celular da Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal Full list of author information is available at the end of the article reassimilation of ammonium released by a number of biochemical processes such as photorespiration, protein catabolism, deamination of amino acids and some specific biosynthetic reactions such as those involving methionine, isoleucine, phenylpropanoid and lignin [1]. Being the first enzyme in the main pathway of ammonium assimilation in higher plants, GS potentially represents a key component of plant nitrogen use efficiency (NUE) and yield and therefore, an extensive number of studies have been dedicated to understand how GS is regulated and how it is involved in the regulation of nitrogen metabolism in plants [1]. Legumes can obtain a significant part of their nitrogen from the atmosphere through a symbiotic interaction with nitrogen fixing bacteria. Perhaps due to this special source of nitrogen, legumes produce protein-rich seeds with a high nutritive value, representing a major source of nutrients for humans and animal livestock. Because of the ecological, nutritional and economic importance of legume seeds, the biochemical and molecular processes underlying their development have been the focus of much research in recent years. With the development of genomic resources for Medicago truncatula, recently this model legume has been chosen for an integrative approach toward understanding seed physiology and great advances have been made in understanding the metabolic control of seed filling and the regulatory network underlying reserve accumulation [2-5]. An original finding raised by these studies is that the genes involved in amino acid metabolism are among the most highly regulated in the seeds of M. truncatula [3]. The accumulation of storage proteins in seeds, involves N-remobilization from vegetative organs, a process in which GS is likely a key regulator, but although the enzyme has been thoroughly investigated in several organs of different plant species, and especially in legumes, it has been poorly investigated in seeds. A number of GS isoenzymes has been identified in plants and classified according to the sub cellular localization as cytosolic (GS1) and plastidic (GS2), which are assumed to play non-overlapping roles. GS1 isoenzymes are involved in nitrogen assimilation and recycling derived from several different physiological processes (reviewed in [6]), whereas GS2 has been mainly implicated in the reassimilation of the ammonia released during photorespiration [7,8]. Genetic studies revealed that GS2 is encoded by a single gene per haploid genome, whereas several genes encode cytosolic polypeptides [9-16]. Functional GS2 allelic genes have been reported in the amphidiploid tobacco [17], the tetraploid alfalfa [18] and the hexaploid wheat [19]. There is no evidence for the existence of multiple GS2 genes in any plant species and clearly, Arabidopsis thaliana and rice, two plants whose genomes have been fully sequenced, contain a single gene encoding GS2. M. truncatula provides an excellent model system for the study of GS, as it contains a very small GS gene family. Only three expressed GS genes have been reported: MtGS1a and MtGS1b encoding cytosolic polypeptides and MtGS2 encoding a plastid located enzyme [12,20,21]. However the genome of M. truncatula has been reported to contain two additional genes related to GS, the pseudogene MtGS1c which was found not to be expressed [21] and a prokaryotic type GSI-like gene, whose function is unknown [22]. This study reports the existence of a new gene encoding a second plastid located isoenzyme in Medicago truncatula. The gene was identified following the development of the genomic resources for M. truncatula [23] and is (...truncated)