Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin

Glutamine synthetase (GS) is the main enzyme involved in ammonia assimilation in plants and is the target of phosphinothricin (PPT), an herbicide commonly used for weed control in agriculture. As a result of the inhibition of GS, PPT also blocks photorespiration, resulting in the depletion of leaf amino acid pools leading to the plant death. Hybrid transgenic poplar (Populus tremula x P. alba INRA clone 7171-B4) overexpressing cytosolic GS is characterized by enhanced vegetative growth [Gallardo, F., Fu, J., Cantón, F.R., García-Gutiérrez, A., Cánovas, F.M., Kirby, E.G., 1999. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210, 19-26; Fu, J., Sampalo, R., Gallardo, F., Cánovas, F.M., Kirby, E.G., 2003. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant Cell Environ. 26, 411-418; Jing, Z.P., Gallardo, F., Pascual, M.B., Sampalo, R., Romero, J., Torres de Navarra, A., Cánovas, F.M., 2004. Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase. New Phytol. 164, 137-145], increased photosynthetic and photorespiratory capacities [El-Khatib, R.T., Hamerlynck, E.P., Gallardo, F., Kirby, E.G., 2004. Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiol. 24, 729-736], enhanced tolerance to water stress (El-Khatib et al., 2004), and enhanced nitrogen use efficiency [Man, H.-M., Boriel, R., El-Khatib, R.T., Kirby, E.G., 2005. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167, 31-39]. In vitro plantlets of GS transgenic poplar exhibited enhanced resistance to PPT when compared with non-transgenic controls. After 30 days exposure to PPT at an equivalent dose of 275 g ha(-1), growth of GS transgenic poplar plantlets was 5-fold greater than controls. The response of young leaves to PPT treatment depends on physiological state as indicated by GS and Rubisco (LSU) levels. Young leaves from control plants, typically in a low differentiation state, respond to the herbicide showing up-regulation of GS and LSU. In contrast, young leaves from transgenic lines, with higher initial GS and LSU levels compared to control, display up-regulation of NADP(+)-isocitrate dehydrogenase. Differences between control and GS transgenics in their response to PPT are discussed in relation to their differences in photosynthetic and photorespiratory capacities (El-Khatib et al., 2004).     

Hairy roots of Brassica napus: I. Applied glutamine overcomes the effect of phosphinothricin treatment

Summary Hairy roots of Brassica napus (rape cv. Giant) were produced by cocultivating leaf and cotyledon explants with Agrobacterium rhizogenes strain A4T. The hairy roots grew prolifically on solid and in liquid media. Incorporation of ammonium sulphate or phosphinothricin (PPT) into the media reduced growth. PPT treatment reduced glutamine synthetase (GS) activity and increased the ammonia content of the hairy roots. We have found that PPT treatment also induces a loss of glutamine from the roots and this may influence root growth. To test this we grew hairy roots in a liquid medium containing 10 mM glutamine. This glutamine treatment overcame the PPT induced suppression of growth but also significantly increased GS activity, reduced ammonia accumulation and increased the levels of glutamate and asparagine.     

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.     

Light-Dependent Ammonium Ion Toxicity of Rice Leaves in Response to Phosphinothricin Treatment

Ammonium ion accumulation in detached rice leaves treated with phosphinothricin (PPT), an inhibitior of glutamine synthetase (GS), was investigated in the light and darkness. PPT treatment increased NH₄ ⁺ content and induced toxicity in rice leaves in the light but not in darkness, suggesting the importance of light in PPT-induced NH₄ ⁺ toxicity in detached rice leaves. PPT treatment in the light resulted in a decrease of activities of the cytosolic form of GS and the chloroplastic form of GS. The photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced NH₄ ⁺ accumulation induced by PPT in the light. In darkness, PPT-induced NH₄ ⁺ accumulation and toxicity were observed in the presence of glucose or sucrose.     

Glutamine synthetase in Scots pine seedlings and its control by blue light and light absorbed by phytochrome

The appearance of glutamine synthetase (GS. EC 6.3.1.2) in response to light and nitrogen (NO ₃ ^- , NH ₄ ⁺ ) was studied in the organs (roots, hypocotyl, cotyledonary whorl) of the Scots pine (Pinus sylvestris L.) seedling. Although GS activity was found to be mainly (> 80%) located in the whorl where it increased strongly in response to light, a significant GS synthesis was also detected in dark-grown seedlings. Anion-exchange chromatography was used to resolve two GS isoforms which appeared to be regulated differentially in the cotyledonary whorls. The isoform (presumably plastidic GS2) which eluted from the column at 90 mM KCl increased drastically in response to light. The other isoform (presumably cytosolic GS1), which eluted at 200 mM KCl, was not stimulated by light but tended to disappear during the experimental period (4 to 12 d after sowing). Immunoblotting of pine extract yielded a prominent band with a molecular weight of 43 kDa. The linear correlation between GS activity and immunodetectable GS protein could be extrapolated through zero, showing that any increase of GS2 activity is to be attributed to the de-novo synthesis of GS protein. Gelfiltration chromatography yielded a molecular mass for the GS holoenzyme of 340 kDa, a value which supports an octameric quarternary structure as previously suggested for angiosperms. While supplying seedlings with 10 mM NO ₃ ^- stimulated GS synthesis in the whorl by 12%, 10 mM NH ₄ ⁺ caused an incipient ammonium toxicity. Experiments using dischromatic light (simultaneous treatment with two light beams to vary the level of the physiologically active form of phytochrome, Pfr, in blue light) revealed that synthesis of GS2 was controlled by light in the same way as previously shown for ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1). Up to 10 d after sowing the strong light effect could be attributed to phytochrome action whereas between 10 and 12 d after sowing phytochrome control of GS-synthesis failed if no blue/ultraviolet-A light was provided. The data show that blue light is required to maintain responsiveness of GS2 synthesis to phytochrome. Both enzymes, GS2 as well as Fd-GOGAT, appear to be regulated coordinately to meet the demands of ammonium assimilation.Abbreviations and Symbols B blue light - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1); - GS glutamine synthetase (EC 6.3.1.2) - R red light - RG9 long-wavelength far-red light defined by the properties of Schott glass filter RG9 - =Pfr/Ptot far-red absorbing form of phytochrome/total phytochrome, wavelength-dependent photoequilibrium of the phytochrome system Research supported by Deutsche Forschungsgemeinschaft (SFB 46 and Schwerpunkt Physiologie der Bäume). We thank J.M. Penther, (Institut für Biologie II, Freiburg, FRG) for his advice on the chromatographic techniques.     

Changes in the activities of chloroplast and cytosolic isoenzymes of glutamine synthetase during normal leaf growth and plastid development in wheat

Soluble protein extracts and chloroplasts from a serial sequence of transverse sections of a 7-d-old wheat leaf (Triticum aestivum cv. Maris Huntsman) were used to study changes in the activity of glutamine synthetase (GS; EC 6.3.1.2) during cell and chloroplast development. Glutamine synthetase activity increased more than 50-fold per cell from the base to the tip of the wheat leaf. Two isoenzymes of GS were separated using fast protein liquid chromatography (FPLC). Glutamine synthetase localized in the cytoplasm (GS1) eluted at about 0.21 M NaCl, and the isoenzyme localized in the chloroplast (GS2) eluted at about 0.33 M NaCl. The increase in GS activity during leaf development was found to be caused primarily by an increase in the activity of the chloroplast GS2. The activity of the cytoplasmic GS1 remained constant as the cells were displaced from the base to the tip of the leaf, whereas GS2 activity increased within the chloroplast throughout development. At the base of the leaf, 26% of total GS activity was cytoplasmic; the remaining 74% was in the chloroplast. At 10 cm from the base, only 4% of the activity was cytoplasmic, and 96% was in the chloroplast. The results indicate that the chloroplast GS2 is probably responsible for most of the ammonia assimilation in the mature wheat leaf, whereas cytoplasmic GS1 may serve a role in immature developing leaf cells.Abbreviations FPLC fast protein liquid chromatography - GS glutamine synthetase - GS1 cytoplasmic glutamine synthetase - GS2 chloroplast glutamine synthetase     

Purification and characterisation of glutamine synthetase fromNocardia corallina

Glutamine synthetase (GS) (EC 6.3.1.2) has been purified 67-fold fromNocardia corallina. The apparentM _r of the GS subunit was approximately 56,000. Assuming the enzyme is a typical dodecamer this indicates a particle mass for the undissociated enzyme of 672,000. The GS is regulated by adenylylation and deadenylylation, and subject to feedback inhibition by alanine and glycine. The pH profiles assayed by the -glutamyl transferase method were similar for NH₄ ⁺-treated and untreated cell extracts and an isoactivity point was not obtained from these curves. GS activity was repressed by (NH₄)₂SO₄ and glutamate. Cells grown in the presence of glutamine, alanine, proline and histidine had enhanced levels of GS activity. The GS ofN. corallina cross-reacted with antisera prepared against GS from a Gram-negativeThiobacillus ferrooxidans strain but not with antisera raised against GS from a Gram-positiveClostridium acetobutylicum strain.     

Nitrogen assimilation and partitioning in two nitrogen-fixing cultivars of Phaseolus vulgaris L.

Two cultivars of Phaseolus vulgaris L., one responsive (Mexico 309) and one less-responsive (Rio Tibagi) to nodulation with Rhizobium were grown in Leonard jars in a greenhouse. Bean plants were either inoculated with a strain of Rhizobium leguminosarum bv. phaseoli (UMR-1899), a vesicular-arbuscular mycorrhizal (VAM) fungus (Glomus etunicatum) or were left non-inoculated (controls). At two harvests (21 and 28 days post-emergence), extracts containing soluble proteins and free amino acids were prepared from leaves, roots and nodules of field beans. Nodulated plants contained a significantly higher concentration of protein and amino acids in all plant parts. Nitrogen-fixing beans invested a significantly greater proportion of total N as protein-N and amino acid-N as compared to VAM or control beans. Abundant nodule-specific proteins (nodulins) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), identified and quantified using scanning densitometry. Rio Tibagi nodules contained a significantly lower concentration of glutamine synthetase (GS) subunits than did Mexico 309 nodules. Glutamate synthase (GOGAT) and GS activities were low relative to other legumes. The transferase/synthetase ratio for GS was also low indicating that the synthetase activity was caturated and was operating at maximal level in these young N₂-fixing associations. Specific nodule activity (SNA) and the level of GS were correlated (r=0.90, p<0.05) for both cultivars at both harvests. GS activity was only 8 or 24% higher than SNA in nodules of Mexico 309 or Rio Tibagi cultivars, respectively, under conditions where substrate was not limiting. This suggests that early in the functioning of this symbiosis N assimilation by GS is the rate-limiting step in N₂ fixation by these two bean cultivars, each with a different symbiotic efficiency. Phaseolus breeding programs that attempt to improve N₂ fixation in beans should identify germplasm that expresses elevated levels of nodule-specific GS or GOGAT, and this material should be used along with effective R. leguminosarum bv. phaseoli strains that have already been selected, to determine superior host-microsymciont associations.     

Nitrogen metabolism of Douglas fir and Scots pine as affected by optimal nutrition and water supply under conditions of relatively high atmospheric nitrogen deposition

Nitrogen metabolism of the needles of 40-year-old Douglas fir and Scots pine trees, growing in two forest stands on cation-poor and acidic sandy soil with a relatively high atmospheric nitrogen deposition was studied. The composition of the free amino acid (FAA) pool, the concentrations of total nitrogen and soluble protein and the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were determined in the needles. An excessive nitrogen supply by a high atmospheric nitrogen deposition in both forest stands was indicated by the high concentrations of total nitrogen and the amino acids arginine, glutamic acid, glutamine and aspartic acid in control trees. In addition the effect of optimal nutrition and water supply (fertigation) on the needle nitrogen metabolism was evaluated. The total concentration of the FAA pool in needles of both tree species was lower in the fertigated than in the non-fertigated (control) trees, except for 1-year-old needles of Scots pine, in which the concentration after fertigation did not differ from the control. The lower total FAA concentration in the fertigated trees could be attributed to arginine, the concentration of which was on average 60% lower than in the control. Neither the concentration of soluble protein nor the activity of GS were influenced by fertigation. The activity of GDH in fertigated trees only differed significantly from the control in October. Scots pine needles had higher concentrations of protein (50%) and higher activities of GS (44%) and GDH (25%) than Douglas fir needles. Possible explanations for the lower vitality of Douglas fir compared to Scots pine are given.     

Intracellular localization of glutamine synthetase in Chlorogloeopsis fritschii

After differential centrifugation of cell-free extracts of Chlorogloeopsis fritschii, 71% of the original glutamine synthetase (GS) activity was associated with the thylakoids, while little activity was detected in the cytoplasmic membranes. Monospecific antiserum to a purified GS inhibited 88% of the enzyme activity in solubilized thylakoid membranes. An antiserum raised against thylakoids gave 81% inhibition. However, using intact thylakoid membranes, only 7% inhibition was obtained with the GS antiserum, indicating that GS is located inside the thylakoid membranes.The author is with the Department of Biological Sciences, University of Science and Technology, Irbid, Jordan     

Activity of the tetramer and octamer of glutamine synthetase isoforms during primary leaf ontogeny of sugar beet (Beta vulgaris L.)

In extracts from the primary leaf blade of sugar beet (Beta vulgaris L.) we separated a chloroplastic isoform (GS 2) of glutamine synthetase (GS, EC 6.3.1.2) and one or two (depending on leaf age) cytosolic isoforms (GS 1a and GS 1b). The latter were prominent in the early (GS 1a) and late stages of leaf ontogeny (GS 1a and GS 1b), whereas during leaf maturation GS 2 was the predominantly active GS isoform. The GS 1 isoforms were active exclusively in the octameric state although tetrameric GS 1 protein was detected immunologically. Their activity stayed at a relatively constant level during leaf ontogeny; an increase was observed only in the senescent leaf. The activity of GS 2, however, changed drastically during primary leaf ontogeny and was modulated by changes in the oligomeric state of the active enzyme. In the early and late stages of leaf ontogeny when GS 2 activity was low (lower than that of the GS 1 isoforms), GS 2 was active only in the octameric state. In the maturing leaf, when GS 2 activity had reached its maximum level (much higher than that of the GS 1 isoforms), 80 of total GS 2 activity was due the activity of the tetrameric form of the enzyme and 20 was due to octameric GS 2. Tetrameric GS 2 was a hetero-tetramer and thus not the unspecific dissociation product of homo-octameric GS 2. In addition, GS 2 activity was modulated by an activation/inactivation of the tetrameric GS 2 protein. Due to an activation of the GS 2 tetramer, the activity of tetrameric GS 2 increased during leaf maturation from zero level 23-fold compared with that of GS 1a and 18-fold compared with that of GS 1b. Possible activators of tetrameric GS 2 are thiol-reactive substances. During leaf senescence, GS 2 activity decreased to zero; this decrease was due to an inactivation of the tetrameric GS 2 protein probably caused by oxidation.Abbreviations FLL final lamina length - FPLC fast protein liquid chromatography - GS glutamine synthetase - GHA -glutamyl hydroxamate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Expression of glutamine-synthetase genes in cotyledons of germinating Phaseolus vulgaris L.

In the legume Phaseolus vulgaris L., glutamine synthetase (GS; EC.6.3.1.2.) is encoded by four actively transcribed genes, gln-, gln-, gln- and gln-. We have studied the expression of these genes in cotyledons during seed germination and have studied the effect of light and nitrate on this process. An RNase-protection method, used to detect the abundances of GS mRNAs, revealed that the four GS genes are differentially expressed in the germinating cotyledons. The gln-. mRNA was present in dry seeds and was the most abundant GS mRNA during early stages of germination. The gln- and gln- mRNAs were first detectable 2 d after sowing and their abundances differed in light- and dark-grown cotyledons at later stages of germination. The gln- mRNA (which encodes the plastid-located GS) was detectable only in light-grown cotyledons, at a low abundance. A nitrate supply of 2 mM had only a minor effect on the expression of the GS genes. Western immunodetection and ion-exchange high-performance liquid chromatography demonstrated that the polypeptide and isoenzyme were present in extracts of dry seeds and represented the major GS products at 2 d and 4 d. Both the and polypeptides appeared at the 2-d stage. The role of differential GS gene expression in controlling cotyledonary GS activity is discussed.Abbreviations 1D, 2D one-, two-dimensional - GS glutamine synthetase - GS_t GS transferase activity - IEX-HPLC ion-exchange high-performance liquid chromatography - kDa kilodaltons - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We are grateful to the Association of Commonwealth Universities and the Science and Engineering Research Council for financially supporting R.S. and to the S.E.R.C. for a grant to support M.J.B. We would like to thank Dr K.J.F. Farnden (University of Otago, New Zealand) and Dr T.H.N. Ellis (John Innes Institute, Norwich) for scanning the autoradiographs for Fig. 2.     

Response of Zea mays to the Inoculation with Azospirillum on Nitrogen Metabolism under Greenhouse Conditions

The maize (Zea mays L.) plants inoculated by N₂-fixing bacterium Azospirillum showed increased activity of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) in root cells free extracts over uninoculated control plants. Maximum differences in NADH-GDH activity were observed during the second and third weeks after sowing. The specific activity of GS showed a greater increase at the end of the assay. The percentage of nitrogen in leaves, root and foliage length, total fresh mass and nitrogenase activity were higher in inoculated plants than in the control ones.