GS Isoforms Research Articles

Cytosolic Glutamine Synthetase GS1;3 Is Involved in Rice Grain Ripening and Germination.

Ammonium is combined with glutamate to form glutamine. This reaction is catalyzed by glutamine synthetase (GS or GLN). Plants harbor several isoforms of cytosolic GS (GS1). Rice GS1;3 is highly expressed in seeds during grain filling and germination, suggesting a unique role in these processes. This study aimed to investigate the role of GS1;3 for rice growth and yield. Tos17 insertion lines for GS1;3 were isolated, and the nitrogen (N), amino acid, and ammonium contents of GS1;3 mutant grains were compared to wild-type grains. The spatiotemporal expression of GS1;3 and the growth and yield of rice plants were evaluated in hydroponic culture and the paddy field. Additionally, the stable isotope of N was used to trace the foliar N flux during grain filling. Results showed that the loss of GS1;3 retarded seed germination. Seeds of GS1;3 mutants accumulated glutamate but did not show a marked change in the level of phytohormones. The expression of GS1;3 was detected at the beginning of germination, with limited promoter activity in seeds. GS1;3 mutants showed a considerably decreased ripening ratio and decreased N efflux in the 12th leaf blade under N deficient conditions. The β-glucuronidase gene expression under control of the GS1;3 promoter was detected in the vascular tissue and aleurone cell layer of developing grains. These data suggest unique physiological roles of GS1;3 in the early stage of seed germination and grain filling under N deficient conditions in rice.

Genome-Wide Characterization of Glutamine Synthetase Family Genes in Cucurbitaceae and Their Potential Roles in Cold Response and Rootstock-Scion Signaling Communication

Glutamine synthetase (GS; EC 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is the key enzyme responsible for the primary assimilation and reassimilation of nitrogen (N) in higher plants. There are two main isoforms of GS in higher plants, classified as cytosolic GS (GS1) and chloroplastic GS (GS2) by their size and subcellular localization. In order to improve the stress tolerance, quality, and yield of cucurbit crops such as cucumbers (Csa, Cucumis sativus L.), pumpkins (Cmo, Cucurbita moschata var. Rifu) are often used as rootstocks. Here, the GS family of the two species were comprehensively analyzed using bioinformatics in terms of aspects of the phylogenic tree, gene structure, chromosome location, subcellular localization, and evolutionary and expression patterns. Seven and four GS gene family members were screened in pumpkin and cucumber, respectively. GS family genes were divided into three groups (one for GS2 and two for GS1) according to their homology and phylogenetic relationships with other species. The analysis of gene ontology annotation of GS family genes, promoter regulatory elements, and tissue-specific expression patterns indicates the potential different biological roles of GS isoforms in Cucurbitaceae. In particular, we have identified a potentially available gene (GS1: CmoCh08G004920) from pumpkin that is relatively highly expressed and tissue-specifically expressed. RT-PCR analysis showed that most CmoGSs are induced by low temperature, and long-term (day 2 to day 9) cold stress has a more obvious effect on the RNA abundance of CmoGS. Our work presents the structure and expression patterns of all candidate members of the pumpkin and cucumber GS gene family, and to the best of our knowledge, this is the first time such work has been presented. It is worth focusing on the candidate genes with strong capacity for improving pumpkin rootstock breeding in order to increase nitrogen-use efficiency in cold conditions, as well as rootstock-scion communication.

Differential expression of multiple glutamine synthetase genes in air-breathing magur catfish, Clarias magur and their induction under hyper-ammonia stress

The present study demonstrates the unique presence of three different gs genes (cmgs01, cmgs02, and cmgs03) in air-breathing ureogenic magur catfish (Clarias magur), which is otherwise reported to be encoded by a single gene in higher vertebrates. Of these three genes, two (cmgs01and cmgs03) were identified as ‘liver’ form, predominantly expressed in liver cells, and the third one as ‘brain’ form (cmgs02), expressed chiefly in brain cells. Molecular characterization studies have revealed conservation of homologous active site residues in all the three gs genes. In silico analysis, accompanied by GS enzyme assay and Western blot analysis of different GS isoforms in different subcellular fractions indicated the mitochondrial localization of cmGS01 and cmGS03 in liver and kidney cells and cytosolic localization of cmGS02 in brain cells. Further, exposure of magur catfish to high external ammonia (HEA; 25 mM NH4Cl) led to a significant induction of multiple gs genes as evidenced by higher expression of different gs mRNAs at variable levels in different tissues. The cmgs01 and cmgs03 mRNA levels elevated significantly in liver, kidney, muscle, and gills, whereas the cmgs02 mRNA level increased considerably in the brain after 14 days of exposure to HEA. These increases in mRNA levels were associated with a significant rise in cmGS01 and cmGS03 proteins in liver, kidney, muscle, and gills, and the cmGS02 protein in the brain after 14 days of exposure to HEA. Therefore, it can be concluded that the unique differential expression of three gs genes and their induction under high ammonia level probably helps in detoxification of ammonia to glutamine and further to urea via the ornithine-urea cycle in ureogenic as well as non-ureogenic tissues of these magur catfish.

Expression characteristics of glutamine synthetase of wheat in Escherichia coli

Glutamine synthetase is a key enzyme in plant nitrogen assimilation. To study the structure of wheat glutamine synthetase isoenzymes, GS1, GSr, GSe, GS2 and GS2p of wheat were cloned into pET-21a, and the expression condition was optimized. Although wheat glutamine synthetase isoenzymes had 70%-80% amino acid sequence homology, the isoforms expressed with different characteristics. Induced at 30 °C, the most expression level of GSr, GSe and GS2 was after 3 h, and of GS1 was at the 7 h whereas no GS2p was expressed, and the GS isoenzymes showed different expression level, with the order of GS1 (22%)>GSr (15%)>GS2 (12%)>GSe (5%). GSe expressed as soluble protein, and GS1 expressed mainly as soluble protein whereas GSr and GS2 expressed as insoluble proteins. Induced at 30 °C for 3 h, mRNA transcript levels of GS isoforms were different, with the order of GSr (7.59)>GS2 (1.84)>GS2p (1.66)>GSe (1.46)>GS1 (1.00). The levels of mRNA transcription were not consistent with the level of the protein translation. The analysis of mRNA secondary structure showed the free energy of translation initiation region of glutamine synthetase isoforms was different, with the order of GS1 (14.4)<GSr (17.2)<GS2 (22.6) <GSe (25.4) <GS2p (31.6), the smaller freed energy, the more unstable mRNA secondary structure of translation initiation region and the higher level of protein expression. Soluble expression condition of glutamine synthetase isozymes was also different, with GS1, GSr, GSe and GS2 induced at 30 °C for 5 h, 16 °C for 15 h, 37 °C for 5 h, and 25 °C for 7 h respectively. The soluble protein showed different expression level with GS1 (20%)>GSr (13%)>GS2 (10%)>GSe (7%), and different activities with GS1>GSe>GS2, and the activity of GSr was not detected. The gene sequence of glutamine synthetase isoenzymes determines the amount, status and activity of proteins expressed in prokaryotic cells.

Differential expression of two galactinol synthase isoforms LcGolS1 and LcGolS2 in developing lentil (Lens culinaris Medik. cv CDC Redberry) seeds

Galactinol synthase (GS, EC 2.4.1.123) catalyzes the transfer of a galactosyl residue from UDP-galactose to myo-inositol to synthesize galactinol, a precursor for raffinose family oligosaccharides (RFO) biosynthesis. Screening, a cDNA library constructed with RNA isolated from developing lentil seeds, with partial GS genes resulted in identification of cDNA clones for two isoforms of GS, LcGolS1 (1336 bp, ORF-1002 bp, 334 amino acids) and LcGolS2 (1324bp, ORF-975bp, 325 amino acids) with predicted molecular weights of 38.7 kDa and 37.6 kDa, respectively. During lentil seed development, LcGolS1 transcripts showed higher accumulation during 26–32 days after flowering (DAF) corresponding to seed desiccation, while LcGolS2 showed maximum accumulation at 24 DAF, prior to increase in LcGolS1 transcripts. GS enzyme activity was maximum at 26 and 28 DAF and corresponded to galactinol accumulation, which also increased rapidly at 22 DAF with maximum accumulation at 26 DAF. Substrates for GS activity, myo-inositol and glucose/galactose were present in high concentrations during early stages of seed development but gradually decreased from 20 DAF to 32 DAF when galactinol concentration increased coinciding with increased GS enzyme activity.

New isoforms and assembly of glutamine synthetase in the leaf of wheat (Triticum aestivum L.).

Glutamine synthetase (GS; EC 6.3.1.2) plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Here, three developmentally regulated isoforms of GS holoenzyme in the leaf of wheat (Triticum aestivum L.) seedlings are described using native-PAGE with a transferase activity assay. The isoforms showed different mobilities in gels, with GSII>GSIII>GSI. The cytosolic GSI was composed of three subunits, GS1, GSr1, and GSr2, with the same molecular weight (39.2kDa), but different pI values. GSI appeared at leaf emergence and was active throughout the leaf lifespan. GSII and GSIII, both located in the chloroplast, were each composed of a single 42.1kDa subunit with different pI values. GSII was active mainly in green leaves, while GSIII showed brief but higher activity in green leaves grown under field conditions. LC-MS/MS experiments revealed that GSII and GSIII have the same amino acid sequence, but GSII has more modification sites. With a modified blue native electrophoresis (BNE) technique and in-gel catalytic activity analysis, only two GS isoforms were observed: one cytosolic and one chloroplastic. Mass calibrations on BNE gels showed that the cytosolic GS1 holoenzyme was ~490kDa and likely a dodecamer, and the chloroplastic GS2 holoenzyme was ~240kDa and likely a hexamer. Our experimental data suggest that the activity of GS isoforms in wheat is regulated by subcellular localization, assembly, and modification to achieve their roles during plant development.

Molecular Docking Analysis Identifies Potential Antagonists of Misregulated Gsα Proteins

McCune-Albright Syndrome (MAS) is a genetic disorder caused by GTPase deficient alleles of Gsα. This disease is characterized by hyperendocrinopathies, skin hyperpigmentation, and polyostotic fibrous dysplasia of bone. While the endocrine disorders can be managed with drugs, the bone disease has no pharmacological treatment, only a path of painful and often unsuccessful orthopedic surgeries. A small molecule inhibitor that is a specific antagonist to the mutant Gs isoform would be of great benefit to MAS patients. Previous work in the lab showed mutations at R231, a surface residue near the GTP-binding site, suppressed the constitutive activity of all MAS-associated mutations in Gs (R201H, R201C, R201S) and partially suppressed the constitutive activity of a Q227L mutation in Gs. Molecules were docked in silico to the R231 region, identifying several molecules with potential to bind to the suppressor site. Transiently transfected HEK cells overexpressing Gs-R201H were treated with each of 18 candidate molecules. Significant reduction in basal cAMP was observed in the presence of either 6-oxo-N-{[2-(pyrrolidin-1-yl)pyridin-4-yl]methyl}-1,6-dihydropyridazine-3-carboxamide or N-{[3-(3-methylphenyl)phenyl]methyl}-6-oxo-1,6-dihydropyridazine-3-carboxamide, while cells transfected with wild-type Gs constructs exhibited no decrease in basal cAMP levels when treated with these compounds. Thus, these compounds may serve as leads for the development of drugs for this rare disease. This work was Supported by NIH grant 1R15ED020190-01

Redundancy and metabolic function of the glutamine synthetase gene family in poplar.

BackgroundGlutamine synthetase (GS; EC: 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is a key enzyme in ammonium assimilation and metabolism in higher plants. In poplar, the GS family is organized in 4 groups of duplicated genes, 3 of which code for cytosolic GS isoforms (GS1.1, GS1.2 and GS1.3) and one group that codes for the choroplastic GS isoform (GS2). Our previous work suggested that GS duplicates may have been retained to increase the amount of enzyme in a particular cell type.ResultsThe current study was conducted to test this hypothesis by developing a more comprehensive understanding of the molecular and biochemical characteristics of the poplar GS isoenzymes and by determinating their kinetic parameters. To obtain further insights into the function of the poplar GS genes, in situ hybridization and laser capture microdissections were conducted in different tissues, and the precise GS gene spatial expression patterns were determined in specific cell/tissue types of the leaves, stems and roots. The molecular and functional analysis of the poplar GS family and the precise localization of the corresponding mRNA in different cell types strongly suggest that the GS isoforms play non-redundant roles in poplar tree biology. Furthermore, our results support the proposal that a function of the duplicated genes in specific cell/tissue types is to increase the abundance of the enzymes.ConclusionTaken together, our results reveal that there is no redundancy in the poplar GS family at the whole plant level but it exists in specific cell types where the two duplicated genes are expressed and their gene expression products have similar metabolic roles. Gene redundancy may contribute to the homeostasis of nitrogen metabolism in functions associated with changes in environmental conditions and developmental stages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0365-5) contains supplementary material, which is available to authorized users.

Constitutive activity of Gs α R201H is suppressed by disruption of either of two hydrophobic pockets within the protein

McCune‐Albright Syndrome (MAS) is a disorder caused by mutations in Gs at R201 that inhibit GTP hydrolysis. We developed a yeast model for MAS using a strain engineered to undergo cell cycle arrest in the presence of constitutively active Gα. We constructed and screened a library of 32,000 additional mutations in a constitutively active Gα gene, and identified 13 potential intragenic suppressor mutations at sites homologous to human Gs. When expressed in HEK cells, three Gs isoforms carrying the MAS (R201H) mutation with one of the 13 substitutions suppressed the MAS mutation's constitutive activity. F142 and L266 participate in hydrophobic packing in the crystal structure. Mutations that reduced the hydrophobicity of either amino acid suppressed the R201H mutation. R231 also suppressed R201H, possibly by altering hydrogen bonds between Switch II and water molecules near the γ phosphate of GTP. The suppressor mutations alone did not alter cellular responses to hormone or constitutively activate the protein. Intragenic suppressor mutations can be used to model potential sites to which small molecule drugs may be targeted to reduce or block the constitutive activity of Gs.Supported by NIH 1R15ED020190–01

Metabolic indicators of drought stress tolerance in wheat: Glutamine synthetase isoenzymes and Rubisco

Drought stress has a considerable impact on the ecosystem and agriculture. Continuous water deficit induces early leaf senescence in plants. During this process, chloroplasts are degraded and photosynthesis drastically drops. The objective of this investigation was to look into the regulation of nitrogen and carbon metabolism during water deficit. Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC4.1.1.39) and the total protein contents inform us of the sink-source relation in plants. Glutamine synthetase (GS, EC 6.3.1.2) isoenzymes are good markers of plastid status (GS2) and the nitrogen metabolism (GS1). Tolerant and sensitive wheat (Triticum aestivum L.) genotypes were tested, which are widely used in agriculture. The amount of protein, Rubisco and GS isoforms in leaves were measured during the grain filling period, as indicative traits that ultimately determine the onset and stage of senescence. The symptoms of senescence first appeared on the oldest and finally on the youngest leaves. Drought stress disrupted the sequentiality of senescence in the sensitive varieties. An untimely senescence appeared in flag leaves, earlier than in the older leaves. Total protein and Rubisco contents decreased and the GS2 isoenzyme declined considerably in the youngest leaves. In the tolerant varieties, however, these physiological parameters did not change under drought, only the sequential senescence of leaf levels accelerated in some cases compared to the control, well-watered plants. Our results revealed that GS is a good indicator of drought stress, which can be applied for the characterization of wheat cultivars in terms of drought stress tolerance.

Regulation of glutamine synthetase isoforms in two differentially drought-tolerant rice (Oryza sativa L.) cultivars under water deficit conditions

KEY MESSAGE : The regulation of GS isoforms by WD was organ specific. Two GS isoforms i.e. OsGS1;1 and OsGS2 were differentially regulated in IR-64 (drought-sensitive) and Khitish (drought-tolerant) cultivars of rice. Water deficit (WD) has adverse effect on rice (Oryza sativa L.) and acclimation requires essential reactions of primary metabolism to continue. Rice plants utilize ammonium as major nitrogen source, which is assimilated into glutamine by the reaction of Glutamine synthetase (GS, EC 6.3.1.2). Rice plants possess one gene (OsGS2) for chloroplastic GS2 and three genes (OsGS1;1, OsGS1;2 and OsGS1;3) for cytosolic GS1. Here, we report the effect of WD on regulation of GS isoforms in drought-sensitive (cv. IR-64) and drought-tolerant (cv. Khitish) rice cultivars. Under WD, total GS activity in root and leaf decreased significantly in IR-64 seedlings in comparison to Khitish seedlings. The reduced GS activity in IR-64 leaf was mainly due to decrease in GS2 activity, which correlated with decrease in corresponding transcript and polypeptide contents. GS1 transcript and polypeptide accumulated in leaf during WD, however, GS1 activity was maintained at a constant level. Total GS activity in stem of both the varieties was insensitive to WD. Among GS1 genes, OsGS1;1 expression was differently regulated by WD in the two rice varieties. Its transcript accumulated more abundantly in IR-64 leaf than in Khitish leaf. Following WD, OsGS1;1 mRNA level in stem and root tissues declined in IR-64 and enhanced in Khitish. A steady OsGS1;2 expression patterns were noted in leaf, stem and root of both the cultivars. Results suggest that OsGS2 and OsGS1;1 expression may contribute to drought tolerance of Khitish cultivar under WD conditions.

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

In higher plants, glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.2) are the predominant enzymes in nitrogen metabolism. In this study, we cloned both the GS and GDH genes and analyzed their expression levels and variations in their activity in developing and germinating x Triticosecale (cv. Witon) kernels. The developing kernel samples were collected 3, 5, 7, 9, 13, 15, 20, 25, 30, 35, 40 and 45 days after flowering (DAF). The germinating kernel samples were collected after 8, 16, 24, 48 and 72 h of imbibition. There are two GS isoforms that are localized to different compartments: the cytosol (GS1) and the chloroplast (GS2). Five cDNAs encoding GS proteins in triticale plants were obtained using RT-PCR. We cloned the four genes encoding GS1, which we designated TsGS1-1, TsGS1-2, TsGS1-3 and TsGS1-4 and the only gene encoding GS2, which was designated TsGS2-1. We studied the changes in the enzymatic activity and the expression profiles of the GDH, GS1 and GS2 genes in both the developing and germinating seeds of triticale. Based on our results, there is likely cooperation between GDH and GS1 in the synthesis of glutamine and glutamate during the early stages of seed formation and in the scutella of kernels for up to 24 h of imbibition.

The glutamine synthetase gene family in Populus.

BackgroundGlutamine synthetase (GS; EC: 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is a key enzyme in ammonium assimilation and metabolism of higher plants. The current work was undertaken to develop a more comprehensive understanding of molecular and biochemical features of GS gene family in poplar, and to characterize the developmental regulation of GS expression in various tissues and at various times during the poplar perennial growth.ResultsThe GS gene family consists of 8 different genes exhibiting all structural and regulatory elements consistent with their roles as functional genes. Our results indicate that the family members are organized in 4 groups of duplicated genes, 3 of which code for cytosolic GS isoforms (GS1) and 1 which codes for the choroplastic GS isoform (GS2). Our analysis shows that Populus trichocarpa is the first plant species in which it was observed the complete GS family duplicated. Detailed expression analyses have revealed specific spatial and seasonal patterns of GS expression in poplar. These data provide insights into the metabolic function of GS isoforms in poplar and pave the way for future functional studies.ConclusionsOur data suggest that GS duplicates could have been retained in order to increase the amount of enzyme in a particular cell type. This possibility could contribute to the homeostasis of nitrogen metabolism in functions associated to changes in glutamine-derived metabolic products. The presence of duplicated GS genes in poplar could also contribute to diversification of the enzymatic properties for a particular GS isoform through the assembly of GS polypeptides into homo oligomeric and/or hetero oligomeric holoenzymes in specific cell types.

Interaction of Cytosolic Glutamine Synthetase of Soybean Root Nodules with the C-terminal Domain of the Symbiosome Membrane Nodulin 26 Aquaglyceroporin

Nodulin 26 (nod26) is a major intrinsic protein that constitutes the major protein component on the symbiosome membrane (SM) of N(2)-fixing soybean nodules. Functionally, nod26 forms a low energy transport pathway for water, osmolytes, and NH(3) across the SM. Besides their transport functions, emerging evidence suggests that high concentrations of major intrinsic proteins on membranes provide interaction and docking targets for various cytosolic proteins. Here it is shown that the C-terminal domain peptide of nod26 interacts with a 40-kDa protein from soybean nodule extracts, which was identified as soybean cytosolic glutamine synthetase GS(1)beta1 by mass spectrometry. Fluorescence spectroscopy assays show that recombinant soybean GS(1)beta1 binds the nod26 C-terminal domain with a 1:1 stoichiometry (K(d) = 266 nm). GS(1)beta1 also binds to isolated SMs, and this binding can be blocked by preincubation with the C-terminal peptide of nod26. In vivo experiments using either a split ubiquitin yeast two-hybrid system or bimolecular fluorescence complementation show that the four cytosolic GS isoforms expressed in soybean nodules interact with full-length nod26. The binding of GS, the principal ammonia assimilatory enzyme, to the conserved C-terminal domain of nod26, a transporter of NH(3), is proposed to promote efficient assimilation of fixed nitrogen, as well as prevent potential ammonia toxicity, by localizing the enzyme to the cytosolic side of the symbiosome membrane.

Ammonium tolerance and the regulation of two cytosolic glutamine synthetases in the roots of sorghum

Tolerance to ammonium nutrition in plants can be related to their ability to detoxify ammonium via nitrogen assimilation in roots. Here, we report that sorghum–sudangrass (Sorghum bicolor L. × S. bicolor var. sudanense) hybrids exhibited enhanced biomass production under high levels of inorganic nitrogen supply as well as increased capacity for nitrogen assimilation in roots. Glutamine synthetase (GS, EC 6.3.1.2) activity and protein accumulated in roots at increasing concentrations of either nitrate or ammonium, with particularly high levels of GS in ammonium-treated plants. Ammonium but not nitrate differentially regulated two distinct cytosolic GS (GS1) isoforms composed by polypeptides of similar size but different charge. The comparative analysis of GS gene sequences and the deduced GS1 polypeptides suggested that the two GS1 isoforms were the expression products of SbGln1.2 and SbGln1.3 genes. SbGln1.3 expression was shown to be upregulated by high levels of inorganic nitrogen supply, with a maximal abundance of SbGln1.3 transcripts in ammonium-grown plants. SbGln1.2 expression was uniform along the root axis meanwhile protein and transcript levels for SbGln1.3 were particularly abundant in the upper part of the axis where lateral roots are prominent. Kinetic analysis revealed that the two GS1 isoenzymes have relatively low-affinity for ammonium ions. The spatial distribution of low-affinity GS1 isoenzymes would provide a sustained glutamine biosynthesis at high levels of ammonium supply and may represent at the same time an efficient system of ammonium detoxification. Such a mechanism may prevent transport of ammonium to leaves alleviating symptoms of toxicity and therefore contributing to sorghum ammonium tolerance.

Control of Liver Glycogen Synthase Activity and Intracellular Distribution by Phosphorylation

Eukaryotic glycogen synthase activity is regulated by reversible phosphorylation at multiple sites. Of the two GS isoforms found in mammals, the muscle enzyme (muscle glycogen synthase) has received more attention and the relative importance of every known phosphorylation site in the control of its activity and intracellular distribution has been previously addressed. We have analyzed the impact of the dephosphorylation at the homologous sites of the glycogen synthase liver (LGS) isoform. Serine residues at these sites were replaced by non-phosphorylatable alanine residues, singly or in pairs, and the resultant LGS variants were expressed in cultured cells using adenoviral vectors. The sole mutation at site 2 (Ser7) yielded an enzyme that was almost fully active and able to induce glycogen deposition in primary hepatocytes incubated in the absence of glucose and in FTO2B cells, a cell line that does not normally synthesize glycogen. Mutation at site 2 was also sufficient to trigger the aggregation and translocation of LGS from the cytoplasm to the hepatocyte cell cortex in the absence of glucose. However, this redistribution was not observed in hepatocytes incubated without glucose when an additional mutation (E509A), which renders the enzyme inactive, was introduced. This result suggests that LGS translocation is strictly dependent on glycogen synthesis.

Glutamine synthetase and glutamate dehydrogenase in cadmium-stressed triticale seedlings

The studies were performed on young triticale seedlings grown on a mineral medium containing 5 mM NO3− as the nitrogen source, with the addition of 0.5 mM CdCl2. It was determined that cadmium ions accumulated mainly in the plant roots. Decreases in nitrate concentrations both in the roots and shoots of seedlings, as well as decreases in soluble protein contents with simultaneous increases in endopeptidase activity were also observed. Both in roots and shoots significant decreases in glutamic acid were noted. Toxic cadmium ion accumulation in seedlings significantly modified activity of primary nitrogen assimilating enzymes, i.e. glutamine synthetase (GS, EC 6.3.1.2) and glutamate dehydrogenase (GDH, EC 1.4.1.2). There was a significant decrease in GS activity both in roots and in shoots of the stressed plants, in comparison to plants grown without cadmium. In shoots of the control plants and plants subjected to stress two GS isoforms were discovered: cytoplasmatic (GS1) and chloroplastic (GS2). Substantial decreases in total glutamine synthetase activity in green parts of seedlings, occurring under stress conditions, result from dramatic decrease in GS2 activity (by 60 % in relation to the control plants); despite simultaneous increases in the cytoplasmatic isoform (GS1) activity by approx. 96 %.

Respective roles of the glutamine synthetase/glutamate synthase cycle and glutamate dehydrogenase in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula.

Our objective was to determine the respective roles of the couple glutamine synthetase/glutamate synthase (GS/GOGAT) and glutamate dehydrogenase (GDH) in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula Gaertn. For this aim, amino acids were analyzed by HPLC and changes in gene expression of several enzymes involved in N and C metabolism were studied by real-time quantitative reverse transcription-polymerase chain reaction. Among the enzymes studied, GDH showed the highest increase in gene expression (80-fold), specifically in the embryo axis and concomitant with the increase in ammonium content during post-germinative growth. In cotyledons, GDH gene expression was very low. Although in vitro GDH aminating activity was several times higher than its deaminating activity, in vivo 15NH4 incorporation into amino acids was completely inhibited by methionine sulfoximine, a GS inhibitor, indicating that GDH is not involved in ammonium assimilation/detoxification. Changes in the expressions of GS and GOGAT isoforms revealed that GS1b (EC 6.3.1.2) in concert with NADH-dependent GOGAT (EC 1.4.1.14) constitute the major route of assimilation of ammonium derived from reserve mobilization and glutamic acid/glutamine synthesis in germinating M. truncatula seeds. However, during post-germinative growth, although germination was held in darkness, expression of GS2 and Fd-GOGAT (EC 1.4.7.1) increased and expression of GS1b decreased in cotyledons but not in the embryo axis. 2-Oxoglutarate, the substrate of the transamination reaction, was provided by the cytosolic isoform of isocitrate dehydrogenase (EC 1.1.1.42). We suggest that GDH during post-germinative growth, specifically in the developing embryo axis, contributes to ammonium delivery to GS for glutamine synthesis in the absence of primary NO3- assimilation. Interestingly, this reaction also produces reducing power (NADH) in organs deprived of photosynthesis.

Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants

ABSTRACTOver‐expression of glutamine synthetase (GS, EC 6.3.1.2), a key enzyme in nitrogen assimilation, may be a reasonable approach to enhance plant nitrogen use efficiency. In this work phenotypic and biochemical characterizations of young transgenic poplars showing ectopic expression of a pine cytosolic GS transgene in photosynthetic tissue (Gallardo et al., Planta 210, 19–26, 1999) are presented. Analysis of 22 independent transgenic lines in a 6 month greenhouse study indicated that expression of the pine GS transgene affects early vegetative growth and leaf morphology. In comparison with non‐transgenic controls, transgenic trees exhibited significantly greater numbers of nodes and leaves (12%), and higher average leaf length and width resulting in an increase in leaf area (25%). Leaf shape was not altered. Transgenic poplars also exhibited increased GS activity (66%), chlorophyll content (33%) and protein content (21%). Plant height was correlated with GS content in young leaves, suggesting that GS can be considered a marker for vegetative growth. Molecular and kinetic characterization of GS isoforms in leaves indicated that poplar GS isoforms are similar to their counterparts in herbaceous plants. A new GS isoenzyme that displayed molecular and kinetic characteristics corresponding to the octomeric pine cytosolic GS1 was identified in the photosynthetic tissues of transgenic poplar leaves. These results indicate that enhanced growth and alterations in biochemistry during early growth are the consequence of transgene expression and assembly of pine GS1 subunits into a new functional holoenzyme in the cytosol of photosynthetic cells.

Aluminum Effects on Nitrogen Uptake and Nitrogen Assimilating Enzymes in Maize Genotypes with Contrasting Tolerance to Aluminum Toxicity

The objective of this study was to investigate the effect of aluminum (Al) on NO3 − and NH4 + uptake and on activity/expression of the N‐assimilating enzymes glutamine synthetase (GS1, GS2, and GSr isoforms, EC 6.3.1.2), glutamate synthase (NADH‐GOGAT, EC 1.4.7.14; ferredoxin‐GOGAT, EC 1.4.7.1), and glutamate dehydrogenase (GDH, EC 1.4.1.2) of maize (Zea mays L.) genotypes contrasting in tolerance to Al toxicity. Seeds were germinated on paper towels, screened for uniformity and allowed to grow for 5–6 d in a modified Steinberg nutrient solution, pH 5.5, in a glasshouse, under natural light. Immediately after transference to a growth chamber (12 h photoperiod, 26°C ± 0.5/18°C ± 0.9 day/night temperature, 72% RH and 540 µ Em−2 s−1 quantum flux density) Al stress was imposed by lowering pH to 4.5 and addition of 166 µmoles L−1 AlCl3. Control plants were grown in the same nutrient solution without Al, pH 4.5. Aluminum inhibited NO3 − and NH4 + uptake and decreased shoot dry matter yield in all genotypes used in this investigation. Irrespective of their tolerance to Al toxicity, Al‐treated plants had less nitrogen (N), phosphorus (P), calcium (Ca), magnesium (Mg), and sulfur (S) in their leaves. In roots, Al treatment did not affect dry matter weight but decreased accumulation of N, Ca, and Mg. Phosphorus and potassium (K) tended to accumulate in roots of Al‐treated plants, suggesting that Al inhibited P translocation to the leaves. In roots, Al induced anaplerotic GDH and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activities, but inhibited GS activity. Moreover, in the apex (0–5 mm) and in the 6–15 mm segment of the elongation zone of most genotypes, Al tended to inhibit expression of GSr and favor expression of GS1. This increase in GS1 expression concomitantly with decreased N uptake and elevated deaminating GDH activity are indicative of metabolic changes usually associated with plant senescence. In leaves, Al did not influence GOGAT and GS activities and expression of GS isoforms suggesting it does not disturb assimilation of nitrogenous compounds translocated from roots to leaves. Nonetheless, lower leaf PEPC activity suggests that Al intoxicated plants had lower CO2 − fixation rates. The inverse effect of Al treatment on the activities of GDH and PEPC in roots and leaves, indicates that whereas increased activities in roots were induced by stress related stimuli, decreased activities in leaves were caused by a reduced nitrogen supply to the leaves. Because the earliest effects observed in this investigation appeared only after 24 h of exposure to Al and were similar in both tolerant and sensitive genotypes, it is concluded that the changes observed in N uptake and assimilation into plant amino acids were downstream of the initial, primary Al toxicity event.