Evidence for a cytosolic-dependent light induction of chloroplastic glutamine synthetase during greening of etiolated rice leaves

During the greening of etiolated rice leaves, total glutamine synthetase activity increases about twofold, and after 48 h the level of activity usually observed in green leaves is obtained. A density-labeling experiment with deuterium demonstrates that the increase in enzyme activity is due to a synthesis of the enzyme. The enhanced activity obtained upon greening is the result of two different phenomena: there is a fivefold increase of chloroplastic glutamine synthetase content accompanied by a concommitant decrease (twofold) of the cytosolic glutamine synthetase. The increase of chloroplastic glutamine synthetase (GS₂) is only inhibited by cycloheximide and not by lincomycin. This result indicates a cytosolic synthesis of GS₂. The synthesis of GS₂ was confirmed by a quantification of the protein by an immunochemical method. It was demonstrated that GS₂ protein content in green leaves is fivefold higher than in etiolated leaves.Abbreviations AbH heavy chain of antibodies - AbL light chain of antibodies - AP acid phosphatase - CH cycloheximide - G6PDH glucose-6-phosphate dehydrogenase - GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ chloroplastic glutamine synthetase - LC lincomycin - NAD-MDH NAD malate dehydrogenase - NADP-G3PDH NADP glyceraldehyde-3-phosphate dehydrogenase     

Senescence-specific increase in cytosolic glutamine synthetase and its mRNA in radish cotyledons

Changes in the levels of cytosolic and chloroplastic isoforms of glutamine synthetase were examined in senescing radish (Raphanus sativus L. cv Comet) cotyledons by immunoblotting analysis using antibodies raised separately against maize glutamine synthetase isoforms. Translatable mRNAs for these isoforms were also examined by analyzing translation products from poly(A)⁺ RNA in a wheat germ system with the antibodies. The relative content of cytosolic isoform (GS₁) increased twofold in the cotyledons that were placed in the dark for 72 hours to accelerate senescence, while that of chloroplastic isoform (GS₂) declined to half of its initial level. The dark-treatment also increased the relative level of translatable mRNA for GS₁ sevenfold after 72 hours, and decreased rapidly that for GS₂ and for other nuclear-coded chloroplast proteins as well. Cotyledons also accumulated GS₁ mRNA when they became senescent after a lengthy growth period under continuous light. These observations suggested that GS₁ genes were activated, while those for GS₂ were repressed, and eventually the population of the enzyme was altered in senescent cotyledonary cells. The role of increased cytosolic enzyme is discussed in relation to the nitrogen metabolism in senescent leaves.     

The Activity and Isoform Complement of Glutamine Synthetase in Panicum Species Differing in Photosynthetic Pathway

Anion exchange chromatography and immunoprecipitation have been used to demonstrate the presence of two forms (GS₁, and GS₂) of glutamine synthetase in the leaves of nine species of Panicum representative of C₃, C₄ and C₃-C₄ intermediate-type photosynthesis. GS₂ from the Panicum species, P. miliaceum and P. maximum was more thermostable than GS₁, GS₁, and GS₂ from P. laxum were equally thermostable but GS₂ from all the Panicum species examined was more sensitive to inhibition by N-ethylmaleimide than GS₁. GS₁, and GS₂ were characterised as being cytoplasmic and chloroplastic isoforms respectively by their reaction with N-ethylmaleimide and by immunoprecipitation with antibodies raised against the cytosolic isoform in barley and the chloroplastic form in tobacco. C₃ species were found to have higher activity of the chloroplastic isoform of glutamine synthetase than C₄ species. C₃-C₄ intermediate species had total leaf glutamine synthetase activities similar to those in C₃ species but were found to have a lower chloroplastic isoform content. The results are consistent with the reassimilation of photorespiratory ammonia by chloroplastic glutamine synthetase.     

Inhibition of plant glutamine synthetases by substituted phosphinothricins

Glutamine synthetase (GS) utilizes various substituted glutamic acids as substrates. We have used this information to design herbicidal α- and γ-substituted analogs of phosphinothricin (l-2-amino-4-(hydroxymethylphosphinyl)butanoic acid, PPT), a naturally occurring GS inhibitor and a potent herbicide. The substituted phosphinothricins inhibit cytosolic sorghum GS₁ and chloroplastic GS₂ competitively versusl-glutamate, with K_i values in the low micromolar range. At higher concentrations, these inhibitors inactivate glutamine synthetase, while dilution restores activity through enzyme-inhibitor dissociation. Herbicidal phosphinothricins exhibit low K_i values and slow enzyme turnover, as described by reactivation characteristics. Both the GS₁ and GS₂ isoforms of plant glutamine synthetase are similarly inhibited by the phosphinothricins, consistent with the broad-spectrum herbicidal activity observed for PPT itself as well as other active compounds in this series.     

Effect of light on the formation of multiple molecular forms of glutamine synthetase in plants

Summary The presence of multiple molecular forms (MMF) of glutamine synthetase (GS) has been studied in pumpkin plants and in cotyledons of bean plants. Two MMF of GS have been found in pumpkin leaves and in green cotyledons: chloroplast GS and cytosol GS. Cotyledons of etiolated pumpkin seedlings contain only the cytosol GS. Illumination of etiolated pumpkin seedlings with white light results in the appearance, within one minute, of the second molecular form, the chloroplast GS, which appears to be due to activation rather than de novo synthesis of the enzyme. Cotyledons of resting seeds of horse bean, pea, soybean and lupine contain only one form of GS. The second form, chloroplast GS, appears after germination in the light, but only in those cotyledons of soybean and lupine that can become green.     

Glutamine Synthetases of Higher Plants : Evidence for a Specific Isoform Content Related to Their Possible Physiological Role and Their Compartmentation within the Leaf

The chromatographic properties of glutamine synthetase isoforms have been investigated in a wide range of higher plant leaves and shoots using ion exchange chromatography. Different patterns of glutamine synthetase isoform content were observed. Among higher plants, four patterns or groups could be recognized. The first group is characterized by having only cytosolic glutamine synthetase, whereas the second group is distinguished by having only chloroplastic glutamine synthetase. The third group is characterized by cytosolic glutamine synthetase being a minor component of the total leaf glutamine synthetase activity. The fourth group is distinct from the other groups in having high cytosolic and chloroplast glutamine synthetase activity. Immunological studies have been undertaken on a few species from each group to identify unambiguously both cytosolic and chloroplastic glutamine synthetases.     

Heterogeneity of Glutamine Synthetase Polypeptides in Phaseolus vulgaris L

Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined.

The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for N₂-fixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides.

     

Induction of nitrate reductase, nitrite reductase, and glutamine synthetase isoforms in sunflower cotyledons as affected by nitrate, light, and plastid integrity

Summary We investigated the inducibility of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), and glutamine synthetase (GS; EC 6.3.1.2) isoforms in cotyledons of 7-day-old seedlings of sunflower (Helianthus annuus L.) in relation to light, nitrogen source (NO ₃ ^– , NO ₂ ^– or NH ₄ ⁺ ), and the involvement of plastids. Nitrate was absolutely (and specifically) required for NR induction, and stimulated more effectively than NO ₂ ^– or NH ₄ ⁺ the synthesis of NiR and chloroplastic GS (GS₂) over the constitutive levels present in N-free-grown seedlings. In vivo inhibition of NR activity by tungsten application to seedlings and measurements of tissue NO ₃ ^– concentration indicate that NO ₃ ^– -dependent enzyme induction is elicited by NO ₃ ^– per se and not by a product of its assimilatory reduction, e.g., NO ₂ ^– or NH ₄ ⁺ . In the presence of NO ₃ ^– , light remarkably enhanced the appearance of NR, NiR, and GS₂, while the activity of the cytosolic GS isoform (GS₁) was adversely affected. Cycloheximide suppressed much more efficiently than chloramphenicol the light- and NO ₃ ^– -dependent increase of GS₂ activity, indicating that sunflower chloroplastic GS is synthesized on cytoplasmic 80S ribosomes. When the plastids were damaged by photooxidation in cotyledons made carotenoid-free by application of norflurazon, the positive action of light and NO ₃ ^– on the appearance of NR, NiR, and GS₂ isoform was greatly abolished. Therefore, it is suggested that intact chloroplasts are required for the inductive effect of light and NO ₃ ^– and/or for the accumulation of newly formed enzymes in the organelle.Abbreviations CAP chloramphenicol - CHX cycloheximide - GS glutamine synthetase - GS₁ cytosolic GS - GS₂ plastidic (chloroplastic) GS - NF norflurazon - NiR nitrite reductase - NR nitrate reductase     

Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase

It is well established that the plastidic isoform of glutamine synthetase (GS₂) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH₄⁺ accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS₂. The mutant plants accumulated high levels of NH₄⁺ when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS₂ that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS₁), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH₄⁺. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS₁ in the mutant, thus revealing a major role of cytosolic GS₁ in the reassimilation and detoxification of photorespiratory NH₄⁺ when the plastidic GS₂ isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity.     

Changes in the cellular localization of cytosolic glutamine synthetase protein in vascular bundles of rice leaves at various stages of development

Cellular localization of cytosolic glutamine synthetase (GS1; EC 6.3.1.2) in vascular bundles of leaf blades of rice (Oryza sativa L.), at the stage at which leaf blades 6 (the lowest position) to 10 were fully expanded, was investigated immunocytologically with an affinity-purified anti-GS1 immunoglobulin G. Strong signals for GS1 protein were detected in companion cells of large vascular bundles when blades 6–8 were tested. Signals for GS1 were also observed in vascular-parenchyma cells of both large and small vascular bundles. The results further support our hypothesis that GS1 is important for the export of leaf nitrogen from senescing leaves. The signals in companion cells were less striking in the younger green leaves and were hardly detected in the non-green portion of the 11th blade. In the non-green blades, strong signals for GS1 protein were detected in sclerenchyma and xylemparenchyma cells. When total GS extracts prepared from the 6th,10th, and the non-green 11th blades were subjected to anion-exchange chromatography, the activity of GS1 was clearly separated from that of chloroplastic GS, indicating that GS1 proteins detected in the vascular tissues were able to synthesize glutamine. The function of GS1 detected in the developing leaves is discussed.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GS1 cytosolic glutamine synthetase - GS2 plastidic glutamine synthetase - IgG immunoglobulin G     

Inorganic nitrogen assimilation in plants of Austrlian rainforest communities

In a study of the plant communities of two Australian rainforests, it was found that pioner species had high levels of nitrate reductase (EC 1.6.6.1) and were predominantly leaf nitrate assimilators. Under- and over-storey species had low levels of shoot and root nitrate reductase activity, and many of them showed little capacity for nitrate reduction even when nitrate ions were freely available. Although closed-forest species have lower levels of nitrate reductase than those of gaps and forest margins, their total nitrogen contents were similar, suggesting the former utilize nitrogen sources other than nitrate ions. Glutamine synthetase (EC 6.3.1.2) was present in the leaves of all species examined. In the leaves of pioneer species the chloroplastic isoform of glutamine synthetase predominted, while in most of the species typical of closed-forest the cytosolic isoform accounted for at least 40% of total leaf activity. Low levels of chloroplastic glutamine synthetase were correlated with a low capacity for leaf nitrate reduction, and both are characteristic of many species that regenerate and grow for some time in shade. Low levels of chloroplastic glutamine synthetase imply that, in some of these woody plants, photorespiratory ammonia is re-assimilated via cytosolic glutamine synthetase.     

Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm,Pinus sylvestris

A full-length cDNA clone (pGSP114) encoding glutamine synthetase was isolated from a gt11 library of the gymnosperm Pinus sylvestris. Nucleotide sequence analysis showed that pGSP114 contains an open reading frame encoding a protein of 357 amino acid residues with a calculated molecular mass of 39.5 kDa. The derived amino acid sequence was more homologous to cytosolic (GS1) (78–82%) than to chloroplastic (GS2) (71–75%) glutamine synthetase in angiosperms. The lack of N-terminal presequence and C-terminal extension which define the primary structure of GS2, also supports that the isolated cDNA encodes cytosolic GS. Southern blot analysis of genomic DNA from P. sylvestris and P. pinaster suggests that GS may be encoded by a small gene family in pine. GS mRNA was more abundant in cotyledons and stems than in roots of both Scots and maritime pines. Western blot analysis in P. sylvestris seedlings showed that only one GS polypeptide, similar in size to GS1 in P. pinaster, could be detected in several different tissues. Our results suggest that cytosolic GS is mainly responsible for glutamine biosynthesis in pine seedlings.This paper is dedicated to the memory of Dr. Jesús S. Olavarría.     

Purification and properties of glutamine synthetase from spinach leaves

The chloroplastic glutamine synthetase of spinach leaves has been purified to homogeneity using affinity chromatography. This involves a tandem `reactive blue A-agarose' and `reactive red-A-agarose' as the final step in the procedure. This procedure results in a yield of 18 milligrams of pure glutamine synthetase per kilogram of starting material. The purity of our enzyme has been demonstrated on both one- and two-dimensional polyacrylamide gels.

Purified glutamine synthetase has a molecular weight of 360,000 daltons and consists of eight 44,000 dalton subunits. The K_m is 6.7 millimolar for glutamate, 1.8 millimolar for ATP (synthetase assay), and 37.6 millimolar for glutamine (transferase assay). The isoelectric point is 6.5 and the pH optima are 7.3 in the synthetase assay and 6.4 in the transferase assay. The irreversible, competitive inhibitors methionine sulfoxamine and phosphinothricin have K_i values of 0.1 millimolar and 6.1 micromolar, respectively. Amino acid analysis has been carried out and the results compared with published analyses for other isoforms of glutamine synthetase.

     

Manipulation of Glutathione and Amino Acid Biosynthesis in the Chloroplast

Poplars (Populus tremula × Populus alba) were transformed to overexpress Escherichia coli γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase in the chloroplast. Five independent lines of each transformant strongly expressed the introduced gene and possessed markedly enhanced activity of the gene product. Glutathione (GSH) contents were unaffected by high chloroplastic glutathione synthetase activity. Enhanced chloroplastic γ-ECS activity markedly increased γ-glutamylcysteine and GSH levels. These effects are similar to those previously observed in poplars overexpressing these enzymes in the cytosol. Similar to cytosolic γ-ECS overexpression, chloroplastic overexpression did not deplete foliar cysteine or methionine pools and did not lead to morphological changes. Light was required for maximal accumulation of GSH in poplars overexpressing γ-ECS in the chloroplast. High chloroplastic, but not cytosolic, γ-ECS activities were accompanied by increases in amino acids synthesized in the chloroplast. We conclude that (a) GSH synthesis can occur in the chloroplast and the cytosol and may be up-regulated in both compartments by increased γ-ECS activity, (b) interactions between GSH synthesis and the pathways supplying the necessary substrates are similar in both compartments, and (c) chloroplastic up-regulation of GSH synthesis is associated with an activating effect on the synthesis of specific amino acids formed in the chloroplast.     

Two genes encoding distinct cytosolic glutamine synthetases are closely linked in the pine genome

The major isoenzyme of glutamine synthetase found in leaves of angiosperms is the chloroplastic form. However, pine seedlings contain two cytosolic glutamine synthetases in green cotyledons: GS1a, the predominant isoform, and GS1b, a minor enzyme whose relative amount is increased following phosphinotricin treatment. We have cloned a GS1b cDNA, and comparison with the previously reported GS1a cDNA sequence indicated that they correspond to separate cytosolic GS genes encoding distinct protein products. Phylogenetic analysis showed that the newly reported sequence is closer to cytosolic angiosperm GS than to GS1a, suggesting therefore that GS1a could be a divergent gymnospermous GS1 gene. Gene mapping using a F2 family of maritime pine showed co-localization of both GS genes on group 2 of the genetic linkage map. This result supports the proposed origin of different members of the GS1 family by adjacent gene duplication. The implications for gymnosperm genome organization are discussed.     

Effects of sodium on mineral nutrition in rose plants

The effects of sodium (Na⁺) ion concentration on shoot elongation, uptake of ammonium (NH₄⁺) and nitrate (NO₃^?) and the activities of nitrate reductase (NR) and glutamine synthetase (GS) were studied in rose plants (Rosa hybrida cv. “Lambada”). The results showed that shoot elongation was negatively correlated with sodium concentration, although no external symptoms of toxicity were observed. Nitrate uptake decreased at high sodium levels, specifically at 30 meq litre4 of sodium. As flower development was normal under high saline conditions, this could suggest that nitrogen was being mobilised from shoot and leaf reserves. Ammonium uptake was not affected by any of the salt treatments applied probably because it diffuses through the cell membrane at low concentrations. Nitrate reductase activity was reduced by 50% at 30 meq litre 1 compared with control treatment, probably due to a decrease in the free nitrate related to nitrate uptake pattern. None of the salt treatments used affected total leaf GS activity (both chloroplastic and cytosolic isoforms) or leaf NPK mineral contents. Nitrate reductase activity in leaves increased at 10 meq litre^?1 of sodium and GS activity in roots (cytosolic isoform only) followed the same pattern as NR. It is suggested that the activation of both enzymes at low salt level could be attributed to the beneficial effect of increased sulphur in the nutrient solutions.     

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia (Rhizobium sp.), newly fixed NH₄⁺ is in equilibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic activity of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in γ-glutamyl transferase assays containing 60 mM Mg²⁺. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of γ-glutamyl transferase activities without and with addition of 60 mM Mg²⁺.Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH₄⁺, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH₄⁺ produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.