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.     

Biochemical and molecular characterization of transgenic<Emphasis Type="Italic"> Lotus japonicus</Emphasis> plants constitutively over-expressing a cytosolic glutamine synthetase gene

Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS₁) or in the chloroplast (GS₂). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS₁ gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS₁ polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22–24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants.Abbreviations GS Glutamine synthetase - UTR Untranslated region     

Expression of glutamine synthetase during the anaerobic germination of Oryza sativa L.

Glutamine synthetase (GS; EC.6.3.1.2.) occurs as cytosolic (GS₁) and plastidic (GS₂) polypeptides. This paper describes the expression of GS isoenzymes in coleoptile during the anaerobic germination of rice (Oryza sativa L.) and the influence of exogenous nitrate on this. By immunoprecipitation with anti-GS serum, two polypeptides of 41- and 44-kDa were detected of which the former was predominant. After fractionation by ion-exchange chromatography, the 41 and 44 kDa bands were identified as GS₁ and GS₂, respectively. Northern blot analysis with specific probes showed the presence of mRNA for cytosolic GS but not for the plastidic form. The presence of exogenous nitrate did not alter the activity and expression of GS in the coleoptile. The role of GS during the anaerobic germination of rice seems to induce the re-assimilation of ammonia rather than the assimilation of nitrate.Abbreviations GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ platidic glutamine synthetase We are grateful to Dr. Julie V. Cullimore for providing GS anti-serum and clones. The research was supported by the National Research Council of Italy, special project RAISA, sub-project N. 2 paper N. 1586.     

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     

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     

Molecular analysis of barley mutants deficient in chloroplast glutamine synthetase

A barley leaf cDNA library has been screened with two oligonucleotide probes designed to hybridize to conserved sequences in glutamine synthetase (GS) genes from higher plants. Two GS cDNA clones were identified as hybridizing strongly to one or both probes. The larger clone (pcHvGS6) contained a 1.6 kb insert which was shown by primer extension analysis to be an almost full-length cDNA. Both clones were more closely related to cDNAs for the chloroplast form of GS (GS₂) from pea and Phaseolus vulgaris than to cDNAs for the cytosolic form (GS₁). A sequence identicalto an N-terminal sequence determined from a purified preparation of the mature GS₂ polypeptide (NH₂-XLGPETTGVIQRMQQ) was found in the pcHvGS6-encoded polypeptide at residues 46–61, indicating a pre-sequence of at least 45 amino acids. The pre-sequence has only limited sequence homology to the pre-sequences of pea and P. vulgaris GS₂ subunits, but is similarly rich in basic residues and possesses some of the structural features common to the targeting sequences of other chloroplast proteins. The molecular lesions responsible for the GS₂-deficient phenotypes of eight photorespiratory mutants of barley were investigated using a gene-specific probe from pcHvGS6 to assay for GS₂ mRNA, and an anti-GS antiserum to assay for GS₂ protein. Three classes of mutants were identified: class I, in which absence of cross-reacting material was correlated with low or undetectable levels of GS₂ mRNA; class II, which had normal or increased levels of GS₂ mRNA but very little GS₂ protein; and class III, which had significant amounts of GS₂ protein but little or no GS₂ activity.     

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 synthetases from rice: purification and preliminary characterization of two forms in leaves and one form in roots

A relatively rapid five-step procedure was used in purifying to apparent homogeneity the glutamine synthetase from roots and one form of the enzyme (GS_I) from leaves of rice. The steps were: preparation of crude extracts, ammonium sulfate precipitation, filtration on Sepharose 4B, fractionation on DEAE-Sephadex A25, and affinity chromatography on ADP-Sepharose 4B. The purified protein appeared as a single band on polyacrylamide gel electrophoresis. Leaf GS_I and the second type of leaf glutamine synthetase (GS_II) formed distinct peaks when eluted from DEAE-Sephadex (step 4). The root enzyme and leaf GS_I were similar in all the properties which were examined. Both enzymes bound to ADP-Sepharose, had similar biosynthetic (18 μmol P/_img protein/min) and transferase (1324 and 1156 μmol γ-glutamyl hydroxamate/mg protein/min) activities, and the same or nearly the same K_m values for glutamate (2.17 mm), Mg²⁺ (4.5 and 5.0 mm), ATP (286 μm), NH₄⁺ (210 and 135 μm), and ADP (3.8 and 5.3 μm). In contrast, leaf GS_II did not bind to ADP-Sepharose and had much higher K_m values for glutamate (8.3 mm), Mg²⁺ (15 mm), NH₄⁺ (684 μm), and ADP (33 μm).     

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.     

Occurrence of Isóenzymes of Glutamine Synthetase in the Alga Chlorella kessleri

Two forms (GS₁ and GS₂) of glutamine synthetase have been isolated, separated by ion exchange chromatography, and partly characterized from cells of the green alga Chlorella kessleri. Both forms are present in cells grown autotrophically or heterotrophically on various nitrogen sources, but under all nutritional conditions GS₁ was found to be the major isoenzyme present (60-80%). The activity of both isoenzymes was greatest in cells grown under nitrogen-limiting conditions. Both isoenzymes have molecular weights in the range 340 to 350,000 daltons. GS₁ was found to have a greater thermostability than GS₂: GS₁ was stable at 30°C while GS₂ lost 95% of its activity in 30 minutes. GS₁ was much less sensitive to thiol reactive reagents than GS₂.     

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.     

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.     

Effect of phosphinothricin (glufosinate) on photosynthesis and photorespiration of C3 and C4 plants

Phosphinothricin (glufosinate), an irreversible inhibitor of glutamine synthetase, causes an inhibition of photosynthesis in C₃ (Sinapis alba) and C₄ (Zea mays) plants under atmospheric conditions (400 ppm CO₂, 21% O₂). This photosynthesis inhibition is proceeding slower in C₄ leaves. Under non-photorespiratory conditions (1000 ppm CO₂, 2% O₂) there is no inhibition of photosynthesis. The inhibition of glutamine synthetase by phosphinothricin results in an accumulation of NH₄ ⁺. The NH₄ ⁺-accumulation is lower in C₄ plants than in C₃ plants. The inhibition of glutamine synthetase through phosphinothricin in mustard leaves results in a decrease in glutamine, glutamate, aspartate, asparagine, serine, and glycine. In contrast to this, a considerable increase in leucine and valine following phosphinothricin treatment is measured. With the addition of either glutamine, glutamate, aspartate, glycine or serine, photosynthesis inhibition by phosphinothricin can be reduced, although the NH₄ ⁺-accumulation is greatly increased. This indicates that NH₄ ⁺-accumulation cannot be the primary cause for photosynthesis inhibition by phosphinothricin. The investigations demonstrate the inhibition of transmination of glyoxylate to glycine in photorespiration through the total lack of amino donors. This could result in a glyoxylate accumulation inhibiting ribulose-1,5-bisphosphate-carboxylase and consequently CO₂-fixation.Abbreviations GOGAT glutamine-2-oxoglutarate-amidotransferase - GS glutamine synthetase - PPT phosphinothricin - MSO methionine sulfoximine - RuBP ribulose-1,5-bisphosphate     

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.     

The value of mutants unable to carry out photorespiration

Manipulation of the CO₂ concentration of the atmosphere allows the selection of photorespiratory mutants from populations of seeds treated with powerful mutagens such as sodium azide. So far, barley lines deficient in activity of phosphoglycolate phosphatase, catalase, the glycine to serine conversion, glutamine synthetase, glutamate synthase, 2-oxoglutarate uptake and serine: glyoxylate aminotransferase have been isolated. In addition one line of pea lacking glutamate synthase activity and one barley line containing reduced levels of Rubisco are available. The characteristics of these mutations are described and compared with similar mutants isolated from populations of Arabidopsis. As yet, no mutant lacking glutamine synthetase activity has been isolated from Arabidopsis and possible reasons for this difference between barley and Arabidopsis are discussed. The value of these mutant plants in the elucidation of the mechanism of photorespiration and its relationships with CO₂ fixation and amino acid metabolism are highlighted.Abbreviations GS cytoplasmic glutamine synthetase - GS₂ chloroplastic glutamine synthetase - PFR Photon fluence rate - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP Ribulose-1,5-bisphosphate - SGAT serine:glyoxylate aminotransferase     

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 NO ₃ ⁻ 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 %. Cadmium ions accumulating in roots and shoots of seedlings not only increased GDH activity, but also modified its coenzymatic specificity.     

The synthesis of pABA: Coupling between the glutamine amidotransferase and aminodeoxychorismate synthase domains of the bifunctional aminodeoxychorismate synthase from Arabidopsis thaliana

Aminodeoxychorismate (ADC) synthase in plants is a bifunctional enzyme containing glutamine amidotransferase (GAT) and ADC synthase (ADCS) domains. The GAT domain releases NH₃ from glutamine and the ADCS domain uses NH₃ to aminate chorismate. This enzyme is involved in folate (vitamin B9) biosynthesis. We produced a stable recombinant GAT–ADCS from Arabidopsis. Its kinetic properties were characterized, and activities and coupling of the two domains assessed. Both domains could operate independently, but not at their optimal capacities. When coupled, the activity of one domain modified the catalytic properties of the other. The GAT activity increased in the presence of chorismate, an activation process that probably involved conformational changes. The ADCS catalytic efficiency was 10⁴ fold higher with glutamine than with NH₄Cl, indicating that NH₃ released from glutamine and used for ADC synthesis did not equilibrate with the external medium. We observed that the GAT activity was always higher than that of ADCS, the excess of NH₃ being released in the external medium. In addition, we observed that ADC accumulation retro-inhibited ADCS activity. Altogether, these results indicate that channeling of NH₃ between the two domains and/or amination of chorismate are the limiting step of the whole process, and that ADC cannot accumulate.     

Carbon and nitrogen metabolism in a barley (Hordeum vulgare L.) mutant with impaired chloroplast dicarboxylate transport

A mutant line, RPr79/2, of barley (Hordeum vulgare L. cv. Maris Mink) has been isolated that has an apparent defect in photorespiratory nitrogen metabolism. The metabolism of ¹⁴C-labelled glutamine, glutamate and 2-oxoglutarate indicates that the mutant has a greatly reduced ability to synthesise glutamate, especially in air, although in-vitro enzyme analysis indicates the presence of wild-type activities of glutamine synthetase (EC 6.3.1.2) glutamate synthase (EC 1.4.7.1 and EC 1.4.1.14) and glutamate dehydrogenase (EC 1.4.1.2). Several characteristics of RPr79/2 are very similar to those described for glutamate-synthase-deficient barley and Arabidopsis thaliana mutants, including the pattern of labelling following fixation of ¹⁴CO₂, and the rapid rise in glutamine content and fall in glutamate in leaves on transfer to air. The CO₂-fixation rate in RPr79/2 declines much more slowly on transfer from 1% O₂ to air than do the rates in glutamate-synthase-deficient plants, and RPr79/2 plants do not die in air unless the temperature and irradiance are high. Analysis of (glutamine+NH₃+2-oxoglutarate)-dependent O₂ evolution by isolated chloroplasts shows that chloroplasts from RPr79/2 require a fivefold greater concentration of 2-oxoglutarate than does the wild-type for maximum activity. The levels of 2-oxoglutarate in illuminated leaves of RPr79/2 in air are sevenfold higher than in Maris Mink. It is suggested that RPr79/2 is defective in chloroplast dicarboxylate transport.