A revised view on the evolution of glutamine synthetase isoenzymes in plants

SUMMARY 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 genes 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) as a result of 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 new view of GS evolution in plants, including a new cytosolic GS group (GS1a), has important functional implications in the context of plant metabolism adaptation to global changes.


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Glutamine synthetase (GS, EC 6.3.1.2) catalyzes the incorporation of ammonium into glutamate 2 0 using ATP to produce glutamine while releasing Pi and ADP (Heldt and Piechulla, 2011). GS is an 2 1 enzyme of major importance, as it represents the main, if not the only, mechanism incorporating 2 2 inorganic nitrogen (N) into organic molecules in virtually all living organisms (Shatters et al. 1989).

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It has been suggested that the genes encoding GS are not only one of the oldest genes in the  Three GS superfamilies have been identified, namely, GSI, GSII and GSIII, with the corresponding  in all three organs. In the needles and in the roots, its expression level was significantly higher only 1 8 7 during the light-dark transition. In the stem, the PpGS1b expression level was the highest when the  In G. biloba seedlings, GbGS1a, GbGS2 and GbGS1b (1 to 3) expression levels were quantified ( Figure 4B). The amount of GbGS2 transcripts was very low both in the stems and roots when the 1 9 5 seedlings were grown under L/D or continuous darkness conditions. In contrast, under these two 1 9 6 conditions, the GbGS2 expression level was at least 20-fold higher in the leaves. Although the 1 9 7 amount of GbGS1a transcripts was higher in the leaves than in the other organs, it was four times 1 9 8 lower than that of GbGS2. The three genes encoding GbGS1b were expressed at a higher level in 1 9 9 the stems and roots than in the leaves. Two significant correlations were found: between the 2 0 0 expression levels of GbGS1a and GbGS2 (0.9) and those of GbGS1b.1 and GbGS1b.2 (0.89). to that of GbGS1a, although the transcript accumulation was three times lower. Transcripts for 2 0 4 GbGS1b.3 were not detected irrespective of the light/dark regime ( Figure 4B). M. grandiflora seedlings were also exposed to different light treatments to study the GS gene MgGS1a exhibited a similar pattern of transcript accumulation, except that for the former, there was 2 1 2 a significant decrease in the light-dark treatment and an increase during the transfer from dark to genes encoding GS. Its pattern of expression in the different organs was similar to that of MgGS2.  stem, in which it was much higher during the L/D cycle. As shown in Figure 5, only four significant  In all plant species, each GS isoenzyme plays a key role either in primary N assimilation or N 2 2 5 recycling, as most of the N-containing molecules required for growth and development are derived 2 2 6 from glutamine, the product of the reaction catalyzed by this enzyme. Throughout evolution, such 2 2 7 important metabolic functions are subjected to a high selective pressure, which made GS Viridiplantae GSII. This study was performed using the corresponding gene sequences belonging to   In the present investigation, the resulting phylogenetic analysis agreed with previous studies in 2 3 5 which two main groups of plant GSII encoded by nuclear genes were identified, namely, GSIIb 2 3 6 (GLN2) and GSIIe (GLN1/GS) (Figures 1 and 2). An HGT event from eubacteria was previously proposed as the more parsimonious process for the emergence of the GLN2 group (Tateno et al. signal peptide in all the GLN2 that allows the targeting of the proteins to organelles such as plastids 2 4 0 and mitochondria (Table S1). Interestingly, genes encoding GLN2 were not identified in vascular isoenzymes are involved 1) in the synthesis of the transport of glutamine and derived amino acids most recent genes encoding GS in plants.

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The phylogeny of the ancient Embryophyta clades (Anthocerotophyta, Marchantiophyta and in nonvascular land plants (Figures 1 and 2). Curiously, GLN2 was also found in these three clades, The occurrence of a gene encoding GS1a has never been previously described in ginkgo, basal  Cys residues involved in the redox modulation of GS2 activity (e.g., C306 in Arabidopsis) was  We also observed that this residue was conserved in all the GS1a and GS2 protein sequences second Cys residue is present in GS2 (e.g., C371 in Arabidopsis) and in a number of GLN1 suggests that it was acquired by angiosperms during plant evolution ( Figure S5).

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When the single-event hypothesis is considered, the emergence of GS2 following the loss of GS1a  which, in addition to photorespiratory ammonium reassimilation, is also responsible for the as an inorganic N source, which can be readily assimilated by cytosolic GS in the absence of GS2.

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Concerning the process of GS2 selection, the most likely hypothesis is the duplication of genes 3 4 9 encoding cytosolic GS leading to functional specialization due to changes such as those in the gene promoter and the addition of a sequence encoding a signal peptide used to import the protein into could be at the origin of GS2 (Figures 1, 3-5).

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Finally, we were able to conclude that the group represented by GS1b evolved in a different way 3 5 5 than that grouping GS1a and GS2. In ginkgo and angiosperms, GS1b is generally represented by a  The combined phylogenetic analysis and gene expression study presented in this work allowed us to names and species for the different GS sequences are presented in Table S1. All nucleotide  find the best fit model among 24 models used to study molecular evolution (Nylander, 2004). The  inference. The initial tree was constructed using the NJ/BioNJ method. The phylogeny test was 4 2 1 performed using the Bootstrap method with 1,000 replications.  Ginkgo, pine, and magnolia seedlings were germinated and grown at 23ºC either with a 16h light/8h    Transcription Supermix (Bio-Rad, Hercules, CA, USA). qPCR was carried out using 10 ng of  The results for maritime pine were normalized using a saposin-like aspartyl protease (unigene1135)   Table S2. Magnolia and ginkgo sequences used to design the primers  Table S3. Rad, Hercules, CA, USA) was used to perform the PCR reaction. Primer sequences were obtained 4 7 1 from C. hainanensis and presented in Table S2. After the initial denaturation step at 98˚C during 1    Choi YA, Kim SG, Kwon YM. 1999. The plastidic glutamine synthetase activity is directly          Evol. Biol.10:198.