Glutamine synthetase (GS) is a key enzyme responsible for the incorporation of inorganic nitrogen in the form of ammonium into the amino acid glutamine. In plants, two groups of functional GS enzymes are found: eubacterial GSIIb (GLN2) and eukaryotic GSIIe (GLN1/GS). Only GLN1/GS genes are found in vascular plants, which suggests that they are involved in the final adaptation of plants to terrestrial life. The present phylogenetic study reclassifies the different GS of seed plants into three clusters: GS1a, GS1b and GS2. The presence of genes encoding GS2 has been expanded to Cycadopsida gymnosperms, which suggests the origin of this gene in a common ancestor of Cycadopsida, Ginkgoopsida and angiosperms. GS1a genes have been identified in all gymnosperms, basal angiosperms and some Magnoliidae species. Previous studies in conifers and the gene expression profiles obtained in ginkgo and magnolia in the present work could explain the absence of GS1a in more recent angiosperm species (e.g., monocots and eudicots) due to the redundant roles of GS1a and GS2 in photosynthetic cells. Altogether, the results provide a better understanding of the evolution of plant GS isoenzymes and their physiological roles, which is valuable for improving crop nitrogen use efficiency and productivity.This work was supported by Spanish Ministerio de Ciencia e Inno-vación grant numbers: BIO2015-73512-JIN MINECO/AEI/FEDER,UE; and RTI2018-094041-B-I00 and EQC2018-004346-P. This work was also supported by Junta de Andalucía, grant number P20_00036 PAIDI 2020/FEDER, UE. Funding for open access charge: Universidad de Málaga/CBUA. JMVM was supported by a grant from the Spanish Ministerio de Educación y Formación Profesional (FPU17/03517). FO was supported by grants from the Universidad de Málaga (Programa Operativo de Empleo Juvenil vía SNJG, UMAJI11, FEDER, FSE, Junta de Andalucía) and BIO-114, Junta de Andalucía
