水稻谷氨酰胺合成酶同工酶免疫学性质比较研究

用纯化的水后（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）根部存在的两种谷氨酰胺合成酶（ＧＳ）同工酶ＧＳｒａ和ＣＳｒｂ分别免疫兔子,得到相应的抗体。免疫扩散和免疫印迹实验表明,ＣＳｒａ、ＧＳｒｂ的抗体对ＧＳ及其同工酶是的。免疫沉淀试验表明,ＧＳｒａ、ＧＳｒｂ不仅识别它的相应的抗原,而且也能很好地识别彼此的抗原。这两种抗体也能较好地识别水稻叶片胞液型的ＧＳＴ,但对水稻叶片和菠菜（Ｓｐｉｎａｃｉａ ｏｌｅｒａｃｅ     

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     

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     

Cellular compartmentation of ammonium assimilation in rice and barley

This review describes immunolocalization studies of the tissue and cellular location of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (Fd GOGAT; EC 1.4.7.1 and NADH-GOGAT; EC 1.4.1.14) proteins in roots and leaves of rice (Oryza sativa L.) and barley (Hordeum vulgare L.). In rice, cytosolic GS (GS1) protein was distributed homogeneously through all cells of the root. NADH GOGAT protein was strongly induced and its cellular location altered by ammonium treatment, becoming concentrated within the epidermal and exodermal cells. Fd GOGAT protein location changed with root development, from a widespread distribution in young cells to becoming concentrated within the central cylinder as cells matured. Plastid GS protein was barely detectable in rice roots, but was the major isoform in leaves, being present in the mesophyll and parenchyma sheath cells. GS1 was specific to the vascular bundle, as was NADH GOGAT, whereas Fd GOGAT was primarily found in mesophyll cells. In barley roots, GS1 protein was found in the cortical and vascular parenchyma and its concentration was highest in N-deficient seedlings. Plastid GS protein was detected in both cortical and vascular cells, where different plastid forms, containing different concentrations of GS protein, were identified. In barley leaves, GS2 protein was detected in the mesophyll chloroplasts and GS1 was found in the mesophyll and vascular cells. N nutrition strongly influenced this distribution, with a marked increase in GS1 concentration in the vascular cells in response to nitrate and ammonium, and an increase in mesophyll GS2 concentration in nitrate-grown seedlings. Fd GOGAT protein was found in both the mesophyll and vascular plastids. These localization studies show that the GS/GOGAT cycle is highly compartmentalized at both the subcellular and cellular levels. Reasons for this compartmentation, and the roles of each isoform, are discussed.     

Post-translational regulation of cytosolic glutamine synthetase by reversible phosphorylation and 14-3-3 protein interaction

Regulation of the cytosolic isozyme of glutamine synthetase (GS(1); EC 6.3.1.2) was studied in leaves of Brassica napus L. Expression and immunodetection studies showed that GS(1) was the only active GS isozyme in senescing leaves. By use of [gamma-(32)P]ATP followed by immunodetection, it was shown that GS(1) is a phospho-protein. GS(1) is regulated post-translationally by reversible phosphorylation catalysed by protein kinases and microcystin-sensitive serine/threonine protein phosphatases. Dephosphorylated GS(1) is much more susceptible to degradation than the phosphorylated form. The phosphorylation status of GS(1) changes during light/dark transitions and depends in vitro on the ATP/AMP ratio. Phosphorylated GS(1) interacts with 14-3-3 proteins as verified by two different methods: a His-tag 14-3-3 protein column affinity method combined with immunodetection, and a far-Western method with overlay of 14-3-3-GFP. The degree of interaction with 14-3-3-proteins could be modified in vitro by decreasing or increasing the phosphorylation status of GS(1). Thus, the results demonstrate that 14-3-3 protein is an activator molecule of cytosolic GS and provide the first evidence of a protein involved in the activation of plant cytosolic GS. The role of post-translational regulation of cytosolic GS and interactions between phosphorylated cytosolic GS and 14-3-3 proteins in senescing leaves is discussed in relation to nitrogen remobilization.     

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine     

Glutamine Synthetase in Rice: A COMPARATIVE STUDY OF THE ENZYMES FROM ROOTS AND LEAVES

Chromatographic, kinetic, and regulatory properties of glutamine synthetase in rice were investigated. By DEAE-Sephacel column chromatography, two forms (glutamine synthetase 1 and glutamine synthetase 2) were identified in leaves and one form (glutamine synthetase R) was identified in roots. Purification on hydroxyapatite and gel electrophoresis showed that glutamine synthetase R was distinct from the leaf enzymes. The three isoforms were purified to similar specific activities and their properties were studied. Heat lability, pH optimum about 8, K(m) for l-glutamate of 20 millimolar, and inhibition by glucosamine 6-phosphate were the main characteristics of glutamine synthetase 2. Heat stability, pH optimum about 7.5, K(m) for l-glutamate of 2 millimolar, and no effect of glucosamine 6-phosphate differentiated glutamine synthetase 1 from glutamine synthetase 2. Glutamine synthetase R was also a labile protein but its kinetic and regulatory properties were quite similar to those of glutamine synthetase 1. These results clearly demonstrate the existence of three isoforms of glutamine synthetase in rice, two of which are located in the leaves and the third in the roots.     

Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula

It was reported recently that the plastid-located glutamine synthetase (GS2) from Medicago truncatula is regulated by phosphorylation catalysed by a calcium-dependent protein kinase and 14-3-3 interaction. Here it is shown that the two cytosolic GS isoenzymes, GS1a and GS1b, are also regulated by phosphorylation but, in contrast to GS2, GS1 phosphorylation is catalysed by calcium-independent kinase(s) and the phosphorylated enzymes fail to interact with 14-3-3s. Phosphorylation of GS1a occurs at more than one residue and was found to increase the affinity of the enzyme for the substrate glutamate. In vitro phosphorylation assays were used to compare the activity of GS kinase, present in different plant organs, against the three M. truncatula GS isoenzymes. All three GS proteins were phosphorylated by kinases present in leaves, roots, and nodules, but to different extents, suggesting a differential regulation under different metabolic contexts. Cytosolic GS phosphorylation was found to be affected by light in leaves and by active nitrogen fixation in root nodules, whereas GS2 phosphorylation was unaffected by these conditions. Some putative GS-binding phosphoproteins were identified showing both isoenzyme and organ specificity. Two phosphoproteins of 70 and 72 kDa were specifically bound to the cytosolic GS isoenzymes. Interestingly, phosphorylation of these proteins was also influenced by the nitrogen-fixing status of the nodule, suggesting that their phosphorylation and/or binding to GS are related to nitrogen fixation. Taken together, the results presented indicate that GS phosphorylation is modulated by nitrogen fixation in root nodules; these findings open up new possibilities to explore the involvement of this post-translational mechanism in nodule functioning.     

Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves

Tissue localizations of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), chloroplastic GS (GS2), and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in rice (Oryza sativa L.) leaf blades were investigated using a tissue-print immunoblot method with specific antibodies. The cross-sections of mature and senescent leaf blades from middle and basal regions were used for tissue printing. The anti-GS1 antibody, raised against a synthetic 17-residue peptide corresponding to the deduced N-terminal amino acid sequence of rice GS1, cross-reacted specifically with native GS1 protein, but not with GS2 after transfer onto a nitrocellulose membrane. Tissue-print immunoblots showed that the GS1 protein was located in large and small vascular bundles in all regions of the leaf blade prepared from either stage of maturity. On the other hand, GS2 and Fd-GOGAT proteins were mainly located in mesophyll cells. The intensity of the developed color on the membrane for GS1 was similar between the two leaf ages, whereas that for GS2 and Fd-GOGAT decreased during senescence. The tissue-specific localization of GS1 suggests that this GS isoform is important in the synthesis of glutamine, which is a major form of nitrogen exported from the senescing leaf in rice plants.     

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.     

Cytosolic localization in tomato mesophyll cells of a novel glutamine synthetase induced in response to bacterial infection or phosphinothricin treatment

In tomato (Lycopersicon esculentum Mill.) leaves, the predominant glutamine synthetase (GS; EC 6.3.1.2) is chloroplastic (GS2; 45 kDa) whereas the cytosolic isoform (GS1; 39 kDa) is represented as a minor enzyme. Following either infection by Pseudomonas syringae pv. tomato (Pst) or treatment with phosphinothricin (PPT), a GS inhibitor, GS1 accumulated in the leaves. In contrast to healthy control leaves, where GS1 was restricted to the veins, in infected and PPT-treated leaves the GS1 polypeptide was also detected in the leaf blade; moreover, it was more abundant than GS2. Different immunological approaches were therefore used to investigate whether or not the GS1 polypeptide expressed in Pst-infected and PPT-treated tomato leaves was distributed among different tissues and subcellular compartments in the same way as the constitutive GS1 expressed in healthy leaves. By tissue-printing analysis, a similar GS immunostaining was observed in epidermis, mesophyll and phloem of leaflet midrib cross-sections of control, infected and PPT-treated leaves. Immunocytochemical localization revealed that GS protein was present in the chloroplast of mesophyll cells and the cytoplasm of phloem cells in healthy leaves; however, in Pst-infected or PPT-treated leaves, a strong labelling was observed in the cytoplasm of mesophyll cells. Two-dimensional analysis of GS polypeptides showed that, in addition to the constitutive GS1, a GS1 polypeptide different in charge was present in tomato leaflets after microbial infection or herbicide treatment. All these results indicate that a novel cytosolic GS is induced in mesophyll cells of Pst-infected or PPT-treated leaves. A possible role for this new cytosolic GS in the remobilization of leaf nitrogen during infection is proposed. Received: 16 January 1998 / Accepted 21 April 1998     

Immunocytolocalization of glutamine synthetase in mesophyll and phloem of leaves ofSolanum tuberosum L.

Summary Localization of glutamine synthetase inSolanum tuberosum leaves was investigated by techniques of Western tissue printing and immunogold electron microscopy. Anti-GS antibodies used in immunolocalization recognize two peptides (45 kDa and 42 kDa) on Western blots. Antibody stained tissue prints on nitrocellulose membranes allowed low resolution localization of GS. Immunostaining was most evident in the adaxial phloem of the leaf midribs and petiole veins. High-resolution localization of glutamine synthetase by immunogold electron microscopy revealed that this enzyme occurs in both the chloroplasts and the cytosol ofS. tuberosum leaf cells. However, GS was specifically associated with the chloroplasts of mesophyll cells and with the cytoplasm of phloem companion cells. The evidence for cell-specific localization of chloroplast and cytosolic GS presented here agrees with the recently reported cell-specific pattern of expression of GUS reporter gene, directed by promoters for chloroplast and cytosolic GS form in tobacco transgenic plants. These data provide additional clues to the interpretation of the functional role of these different isoenzymes and its relationship with their specific localization.Abbreviations BSA bovine serum albumin - EM electron microscope - GOGAT glutamate synthase - GS glutamine synthetase - GUS -glucuronidase - IgG immunoglobulin - PBS phosphate buffer saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Distribution of two isoforms of glutamine synthetase in bundle sheath and mesophyll cells of corn leaves

Localization of two isoforms of glutamine synthetase (GS; EC 6.3.1.2) was investigated in different cell types, mesophyll cells and bundle sheath cells, of corn ( Zea mays L. var. W64A × W182E) leaves by using ion exchange chrotnatography. In whole leaf extracts, relative activities of GS1 (cytosolic GS) and GS2 (chloroplastic GS) were almost equal. Purified mesophyll protoplasts and bundle sheath strands also showed similar proportions of GS1 and GS2. Methionine sulfoximine (1 mM ) enhanced the accumulation of ammonia when mesophyll protoplasts were incubated with nitrite or when bundle sheath strands were incubated with glycine. This clearly indicates a spatial separation of metabolism of NH⁺₄ derived from photorespiration and from reduction of NOJ.     

Glutamine Synthetase in Spinach Leaves : IMMUNOLOGICAL STUDIES AND IMMUNOCYTOCHEMICAL LOCALIZATION

By polyacrylamide gel electrophoresis, DEAE Sephacel, and hydroxyapatite chromatography, one form of glutamine synthetase has been identified in spinach (Spinacia oleracea L. cv. Monstrueux de Viroflay) leaves. It is localized only inside the chloroplast. The enzyme was purified to homogeneity and specific antibodies against the protein were raised by immunization of rabbits. The intracellular localization of glutamine synthetase in spinach leaves was studied by indirect immunofluorescence microscopy on thin-sectioned spinach leaves. It has been demonstrated that the enzyme is specifically associated with the chloroplasts of parenchymatous cells.     

Organ-specific changes in the activity and subunit composition of glutamine-synthetase isoforms of barley (Hordeum vulgare L.) after growth on different levels of NH+ 4

One cytosolic glutamine synthetase (GS, EC 6.3.1.2) isoform (GS 1a) was active in the germinating seeds of barley (Hordeum vulgare L.). A second cytosolic GS isoform (GS 1b) was separated from the leaves as well as the roots of 10-d-old seedlings. The chloroplastic isoform (GS 2) was present and active only in the leaves. The three GS isoforms were active in N-supplied (NH⁺ ₄ or NO ₃ ^– ) as well as in N-free-grown seedlings. This indicates (i) that a supply of nitrogen to the germinating seeds was not necessary for the induction of the GS isoforms and (ii) that no nitrogen-specific isoforms appeared during growth of seedlings with different nitrogen sources. The activity of GS, however, depended on the seedlings' nitrogen source: the specific activity was much higher in the leaves and much lower in the roots of NH⁺ ₄-grown barley than in the respective organs of NO ₃ ^– -fed or N free-grown plants. With increasing concentrations of NH⁺ ₄ (supplied hydroponically during growth), the specific activity of GS 1b increased in the leaves, but decreased in the roots. The activity of GS 2 (leaf) also increased with increasing NH⁺ ₄ supply, whereas GS 1a activity (leaf and root) was not affected. The changes in the activities of GS 1b and GS 2 were correlated with changes in the subunit compositions of the active holoenzymes: growth at increased levels of external NH⁺ ₄ resulted in an increased abundance of one of the four GS subunits, and of two of the five GS 1b subunits in the leaves. In the roots, however, the abundance of these two GS 1b subunits was decreased under the same growth conditions, indicating an organ-specific difference either in the expression of the genes coding for the respective GS 1b subunits or in the assembly of the GS 1b holoenzymes. Furthermore, growth at different levels of NH⁺ ₄ resulted in changes in the substrate affinities of the isoforms GS 1b (root and leaf) and GS 2 (leaf), presumably due to the changes in the subunit compositions of the active holoenzymes.Abbreviations FPLC fast protein liquid chromatography - GHA -glutamyl hydroxamate - GS glutamine synthetase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

菠菜叶中存在两种谷氨酰胺合成酶同工酶

运用非变性聚丙稀酰胺凝胶电泳结合活性染色的方法,在菠菜(Spinacia oleracea L．)生长发育过程中,观察到叶片中至少存在2种谷氨酰胺合成酶(GS),其中一种GS的活性随发育进程而逐渐升高,而另一种GS的活性逐渐降低。在不同来源的成熟的菠菜叶片中同样观察到2种GS的存在。