Glutamine-synthetase isoforms appearing in sunflower cotyledons during germination

Ion-exchange chromatography has been used to separate the isoforms of glutamine synthetase (GS; EC 6.3.1.2) appearing in sunflower (Helianthus annuus L. cv. Peredovic) cotyledons during seedling growth under different light and nitrogen conditions. Both in dry and imbibed seeds, only a single form of GS (GS_s) was detected. Upon seed germination, the GS_s isoform was gradually replaced by cytosolic (GS₁) and plastidic (GS₂) isoforms. Light and nitrate decreased the levels of GS₁. In contrast, the appearance of GS₂ was greatly stimulated by light. Nitrate also had a positive effect, particularly in the light. Light and nitrate acted synergistically on the appearance of GS₂. The GS₂:GS₁ ratio in cotyledons of 9-d-old seedlings ranged from about 2, in darkness and nitrate-deprivation conditions, to 16 under light and nitrate application. The possible physiological roles of the distinct GS isoforms appearing in the epigeal cotyledons of sunflower during germination, and their differential regulation by light and nitrate, are discussed.Abbreviations GS glutamine synthetase - GS₁ cytosolic GS - GS₂ plastidic GS - GS_s GS from seeds This work was supported by a grant from Dirección General de Investigatión Científica y Técnica (PB90-0777) and Plan Andaluz de Investigación (3261), Spain. P.C. gratefully acknowledges receipt of a scholarship from Junta de Andalucía. The valuable technical assistance of Mrs. G. Alcalá is greatly appreciated. We are also grateful to Eurosemillas (Córdoba) for supplying us with sunflower seeds.     

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     

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     

Glutamine synthetase of Chlamydomonas: its role in the control of nitrate assimilation

Work is described which suggests that glutamine synthetase (GS) could play an important and direct regulatory role in the control of NO₃ assimilation by the alga. In both steady-state cells and ones disturbed physiologically by changes in light or nitrogen supply the assimilation of NO₃ appears to be limited by the activity of GS. Moreover although in normal cells NH₃ can completely inhibit NO₃ uptake, promote the deactivation of nitrate reductase (NR) and repress the synthesis of NR and nitrite reductase (NIR), these controls are relaxed in cells in which GS is deactivated by treatment with L-methionine-DL-sulfoximine (MSO). It is proposed that the reversible deactivation of GS may play an important part in the regulation of NO₃ assimilation although it is still not clear whether the enzyme itself or products of its metabolism are responsible.Abbreviations GS glutamine synthetase - GS_s glutamine synthetase, synthetase activity - GS_t glutamine synthetase, transferase activity - NR nitrate reductase - NIR nitrite reductase - GDH glutamate dehydrogenase - CHX cycloheximide - MSO L-methionine-DL-sulfoximine - FAD flavine adenine dinucleotide     

Glutamine synthetases of green and etiolated leaves ofSinapis alba

Studies on the glutamine synthetases (GS, EC 6.3.1.2) of green (GS₂) and etiolated leaves (GS_et) ofSinapis alba L. (cv. Steinacher) revealed striking similarities between the respective enzyme proteins. The enzymes showed corresponding chromatographic properties, both on dimethylaminoethyl-Sephacel and on hydroxylapatite columns. The purified GS proteins were also identical with regard to the molecular weight of their subunits. Isoelectrofocusing of pure GS_et yielded two distinct polypeptide bands in the pH 5.6 region of the gels. This pattern corresponded to the two strong bands of GS₂. Two charge variants of GS polypeptides could be detected by Western-blot analysis of the soluble protein of green leaves using antibodies against mustard GS₂. In immunoprecipitation experiments, the holoenzymes of GS₂ and GS_et were recognized with identical affinities by this antiserum. We conclude that strong similarities exist between the proteins of the GS enzymes in green and etiolated leaves of mustard. Most probably only one GS form, namely the plastidic enzyme, can be found in the epigeal organs ofSinapis. The polypeptides of the GS₂ subunits showed no differences in the hydrophobicity of the polypeptide chains. Neither glucosyl nor mannosyl residues could be detected. Dedicated to Professor Dr. H. Mohr on the occasion of his 60th birthday     

Glutamine synthetase isoenzymes of Phaseolus vulgaris L.: subunit composition in developing root nodules and plumules

In the legume Phaseolus vulgaris L., glutamine synthetase (GS) (EC.6.3.1.2.) occurs as three cytosolic polypeptides, , and , and a plastidic polypeptide, . This paper describes the subunit composition of active octameric GS isoenzymes from root nodules and plumules using ionexchange high-performance liquid chromatography followed by two-dimensional denaturing gel electrophoresis and Western immunodetection. Root nodules contained four separable GS activities, three of which were composed mainly of cytosolic , / and GS polypeptides, whereas the fourth activity, consisted of plastidic GS polypeptides. The increase in GS activity during nodulation was due largely to the appearance of -containing isoenzymes, and to a lesser extent on the isoenzyme, whereas the -isoenzyme activity remained approximately constant throughout. Plumule GS from imbibed seeds was found to be composed of separate and isoenzymes, but 2 d after germination, plumule GS consisted of a mixture of , / and isoenzymes. The results from both nodules and plumules indicate that different cytosolic GS polypeptides in P. vulgaris are able to assemble into both homo-octameric and heterooctameric isoenzymes. Moreover, the changes in the patterns of isoenzymes observed during nodule development and plumule growth are interpreted to be caused both by temporal changes in the denovo synthesis of the polypeptides and also by their spatial separation in different cell types.Abbreviations 1D, 2D one-, two-dimensional - GS glutamine synthetase - GS_s GS semibiosynthetic activity - GS_t GS transferase activity - IEX-HPLC ion-exchange high-performance liquid chromatography - kDa kilodaltons - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Nitrate assimilation during the anaerobic germination of rice: expression of ferredoxin-dependent glutamate synthase

Ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) is the last enzyme involved in the pathway of nitrate assimilation in higher plants. This paper describes the synthesis and expression of the enzyme in anaerobic coleoptiles of rice (Oryza sativa L.) and its regulation by exogenous nitrate. The activity of Fd-GOGAT was strongly inhibited by cycloheximide between 4 and 9 d of anaerobic germination. The addition of nitrate slightly increased, in the first 5 h, the specific activity of Fd-GOGAT as well as the amount of a 160-kDa protein specifically immunoprecipitated with anti-Fd-GOGAT serum. Northern blot analysis, performed with a specific riboprobe, showed the presence of mRNA of the expected size and the inductive effect of nitrate. The role of Fd-GOGAT is discussed in relation to the anaerobic assimilation of nitrate by rice coleoptiles.Abbreviations CHX cycloheximide - Fd ferredoxin - GOGAT glutamate synthase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase The authors wish to thank Dr. J. Turner (Rothamsted Experimental Station, Harpenden, UK) for providing Fd-GOGAT antibody and Dr. H. Sakakibara (Nagoya University, Nagoya, Japan) for Fd-GOGAT clone. This research was supported by the National Research Council of Italy, special project RAISA, sub-projekt N. 2, paper N. 2174.     

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.     

Appearance of a novel form of plant glutamine synthetase during nodule development in Phaseolus vulgaris L.

The activities of glutamine synthetase (GS), nitrogenase and leghaemoglobin were measured during nodule development in Phaseolus vulgaris infected with wild-type or two non-fixing (Fix^-) mutants of Rhizobium phaseoli. The large increase in GS activity which was observed during nodulation with the wild-type rhizobial strain occurred concomitantly with the detection and increase in activity of nitrogenase and the amount of leghaemoglobin. Moreover, this increase in GS was found to be due entirely to the appearance of a novel form of the enzyme (GS_n1) in the nodule. The activity of the form (GS_n2) similar to the root enzyme (GS_r) remained constant throughout the experiment. In nodules produced by infection with the two mutant strains of Rhizobium phaseoli (JL15 and JL19) only trace amounts of GS_n1 and leghaemoglobin were detected.Abbreviations DEAE-Sephacel diethylaminoethyl-Sephacel - GS glutamine synthetase     

The chloroplast-located glutamine synthetase of Phaseolus vulgaris L.: nucleotide sequence,expression in different organs and uptake into isolated chloroplasts

Work using a full-length cDNA clone has revealed that the plastid-located glutamine synthetase (GS) of Phaseolus vulgaris is encoded by a single nuclear gene. Nucleotide sequencing has shown that this cDNA is more closely related to a cDNA encoding the plastidic GS of Pisum sativum than to cDNAs encoding three different cytosolic GS subunits of P. vulgaris. The plastid GS subunits are initially synthesized as higher M _r (47000) precursors containing an N-terminal presequence of about 50 amino acids which is structurally similar to the presequences of other nuclear-encoded chloroplast proteins. The precursor has been synthesized in vitro and is imported by isolated pea chloroplasts and processed to two polypeptides of the same size as native P. vulgaris chloroplast GS subunits (M _r 42000). Experiments with fusion proteins show that the N-terminal 68 amino acids of this precursor allow the cytosolic GS subunit also to be imported and processed by isolated chloroplasts. Polyadenylated mRNA specifically related to the plastidic GS gene is most highly abundant in chloroplast-containing organs (leaves and stems) but is also detectable in roots and nodules.     

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     

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.     

Seasonal variation of leaf glutamine synthetase isoforms in temperate deciduous trees strongly suggests different functions for the enzymes

Changes in the activities of leaf glutamine synthetase (GS) isoforms were followed in four temperate deciduous trees from full leaf expansion to senescence (May to November). In the early part of the season, total GS activity was high in all species, with values ranging from 90 to 200 μmol h⁻¹ g⁻¹ fw. During this early period this activity comprised only the activity of the chloroplastic (GS₂) isoform in all species. These high GS₂ activities are consistent with the role of GS₂ in the re-assimilation of photorespired ammonia. The early high values also coincided with high nitrate reductase activity in one of the species, the highly nitrophilous species Sambucus nigra, with values of up to 16μmol h⁻¹ g⁻¹ fw. This indicates that GS₂ is also important in the assimilation of ammonia produced from nitrate reduction. From mid- to late-season, the cytosolic isoform (GS₁) was detected in all four species and became increasingly more active in comparison to GS₂. By the time of senescence it was the dominant enzyme of the two forms in both S. nigra and Carpinus betulus. The results provide strong support for recent findings that GS₁ is an important enzyme for the mobilization of nitrogen for translocation or storage.     

Purification and Characterization of Two Forms of Glutamine Synthetase from the Pedicel Region of Maize (Zea mays L.) Kernels

Maize (Zea mays L.) kernel pedicels, including vascular tissues, pedicel parenchyma, placento-chalazal tissue, and the surrounding pericarp, contained two forms of glutamine synthetase (EC 6.3.1.2), separable by anion exchange chromatography under mildly acidic conditions. The earlier-eluting activity (GS_p1), but not the later-eluting activity (GS_p2), was chromatographically distinct from the maize leaf and root glutamine synthetases. The level of GS_p1 activity changed in a developmentally dependent manner while GS_p2 activity was constitutive. GS_p1 and GS_p2 exhibited distinct ratios of transferase to hydroxylamine-dependent synthetase activities (5 and 23, respectively), which did not change with kernel age. Purified pedicel glutamine synthetases had native relative molecular masses of 340,000, while the subunit relative molecular masses differed slightly at 38,900 and 40,500 for GS_p1 and GS_p2, respectively. Both GS forms required free Mg²⁺ with apparent K_ms = 2.0 and 0.19 millimolar for GS_p1 and GS_p2, respectively. GS_p1 had an apparent K_m for glutamate of 35 millimolar and exhibited substrate inhibition at glutamate concentrations greater than 90 millimolar. In contrast, GS_p2 exhibited simple Michaelis-Menten kinetics for glutamate with a K_m value of 3.4 millimolar. Both isozymes exhibited positive cooperativity for ammonia, with S_0.5 values of 100 and 45 micromolar, respectively. GS_p1 appears to be a unique, kernel-specific form of plant glutamine synthetase. Possible functions for the pedicel GS isozymes in kernel nitrogen metabolism are discussed.     

Subunit Composition of Glutamine Synthetase Isozymes from Root Nodules of Bean (Phaseolus vulgaris L.)

Glutamine synthetase from bean nodules can be separated into two isoforms, GS_n1 and GS_n2. A purification protocol has been developed. It included protamine sulfate precipitation, ammonium sulfate fractionation, anthranilate-affinity chromatography, Dye-Matrex (Orange A) chromatography, and diethylaminoethyl-cellulose ion-exchange chromatography. GS_n1 and GS_n2 have been purified to homogeneity. Subunit structure analysis using two-dimensional polyacrylamide gel electrophoresis revealed that GS_n1 was composed of two different types of subunit polypeptides. They differed in isoelectric points (6.0 and 6.3) but had the same molecular weights (46,000 Daltons). GS_n2 was composed of only one type of subunit polypeptide. It had an isoelectric point of 6.0 and a molecular weight of 46,000 Daltons. It was apparently identical to one of the polypeptides found in GS_n1. Glutamine synthetase holoenzyme consisted of eight subunits. In the nodule there are two different types of glutamine synthetase subunit polypeptides. Random combinations of the polypeptides should generate nine different isozymes. Our electrophoretic analysis revealed that GS_n2 was but one of the isozymes, and GS_n1 was a composite of the other eight. Hence, nodule glutamine synthetase isozymes were homo-octameric as well as hetero-octameric.     

Development of nitrogen-assimilating enzymes in sunflower cotyledons during germination as affected by the exogenous nitrogen source

Activities of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.3) were measured in cotyledons of sunflower (Helianthus annuus L. cv Peredovic) seedlings during germination and early growth under various external nitrogen sources. The presence of NO ₃ ^- in the medium promoted a gradual increase in the levels of NR and NiR activities during the first 7 d of germination. Neither NR nor NiR activities were increased in a nitrogen-free medium or in media with either NH ₄ ⁺ or urea as nitrogen sources. Moreover, the presence of NH ₄ ⁺ did not abolish the NO ₃ ^- -dependent appearance of NR and NiR activities. The increase of NR activity was impaired both by cycloheximide and chloramphenicol, which indicates that both cytoplasmic 80S and plastidic 70S ribosomes are involved in the synthesis of the NR molecule. By contrast, the appearance of NiR activity was only inhibited by cycloheximide, indicating that NiR seems to be exclusively synthesized on the cytoplasmic 80S ribosomes. Glutamine-synthetase activity was also strongly increased by external NO ₃ ^- but not by NH ₄ ⁺ or urea. The appearance of GS activity was more efficiently suppressed by cycloheximide than chloramphenicol. This indicates that GS is mostly synthesized in the cytoplasm. The cotyledons of the dry seed contain high levels of GDH activity which decline during germination independently of the presence or absence of a nitrogen source. Cycloheximide, but not chloramphenicol, greatly prevented the decrease of GDH activity.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase     

A Role for Glutamine Synthetase in the Remobilization of Leaf Nitrogen during Natural Senescence in Rice Leaves

Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of corresponding mRNAs were determined in leaves of hydroponically grown rice (Oryza sativa) plants during natural senescence. The plants were grown in the greenhouse for 105 days at which time the thirteenth leaf was fully expanded. This was counted as zero time for senescence of the twelfth leaf. The twelfth leaf blade on the main stem was analyzed over a time period of −7 days (98 days after germination) to +42 days (147 days after germination). Total GS activity declined to less than a quarter of its initial level during the senescence for 35 days and this decline was mainly caused by a decrease in the amount of GS2 polypeptide. Immunoblotting analyses showed that contents of other chloroplastic enzymes, such as ribulose-1,5-bisphosphate carboxylase/oxygenase and Fd-glutamate synthase, declined in parallel with GS2. In contrast, the GS1 polypeptide remained constant throughout the senescence period. Translatable mRNA for GS1 increased about fourfold during the senescence for 35 days. During senescence, there was a marked decrease in content of glutamate (to about one-sixth of the zero time value); glutamate is the major form of free amino acid in rice leaves. Glutamine, the major transported amino acid, increased about threefold compared to the early phase of the harvest in the senescing rice leaf blades. These observations suggest that GS1 in senescing leaf blades is responsible for the synthesis of glutamine, which is then transferred to the growing tissues in rice plants.