Changes in the cellular and subcellular localization of glutamine synthetase and glutamate dehydrogenase during flag leaf senescence in wheat (Triticum aestivum L.)

In order to improve our understanding of the regulation of nitrogen assimilation and recycling in wheat (Triticum aestivum L.), we studied the localization of plastidic (GS2) and cytosolic (GS1) glutamine synthetase isoenzymes and of glutamate dehydrogenase (GDH) during natural senescence of the flag leaf and in the stem. In mature flag leaves, large amounts of GS1 were detected in the connections between the mestome sheath cells and the vascular cells, suggesting an active transfer of nitrogen organic molecules within the vascular system in the mature flag leaf. Parallel to leaf senescence, an increase of a GS1 polypeptide (GS1b) was detected in the mesophyll cytosol of senescing leaves, while the GS protein content represented by another polypetide (GS1a) in the phloem companion cells remained practically constant in both leaves and stems. Both GDH aminating activity and protein content were strongly induced in senescing flag leaves. The induction occurred both in the mitochondria and in the cytosol of phloem companion cells, suggesting that the shift in GDH cellular compartmentation is important during leaf nitrogen remobilization although the metabolic or sensing role of the enzyme remains to be elucidated. Taken together, our results suggest that in wheat, nitrogen assimilation and recycling are compartmentalized between the mesophyll and the vasculature, and are shifted in different cellular compartments within these two tissues during the transition of sink leaves to source leaves.     

Glutamine synthetase-glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sink-source nitrogen cycle in tobacco

Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.     

Glutamine synthetase in the phloem plays a major role in controlling proline production

To inhibit expression specifically in the phloem, a 274-bp fragment of a cDNA (Gln1-5) encoding cytosolic glutamine synthetase (GS1) from tobacco was placed in the antisense orientation downstream of the cytosolic Cu/Zn superoxide dismutase promoter of Nicotiana plumbaginifolia. After Agrobacterium-mediated transformation, two transgenic N. tabacum lines exhibiting reduced levels of GS1 mRNA and GS activity in midribs, stems, and roots were obtained. Immunogold labeling experiments allowed us to verify that the GS protein content was markedly decreased in the phloem companion cells of transformed plants. Moreover, a general decrease in proline content in the transgenic plants in comparison with wild-type tobacco was observed when plants were forced to assimilate large amounts of ammonium. In contrast, no major changes in the concentration of amino acids used for nitrogen transport were apparent. A (15)NH(4)(+)-labeling kinetic over a 48-hr period confirmed that in leaves of transgenic plants, the decrease in proline production was directly related to glutamine availability. After 2 weeks of salt treatment, the transgenic plants had a pronounced stress phenotype, consisting of wilting and bleaching in the older leaves. We conclude that GS in the phloem plays a major role in regulating proline production consistent with the function of proline as a nitrogen source and as a key metabolite synthesized in response to water stress.     

Transport and metabolism of raffinose family oligosaccharides in transgenic potato

Raffinose family oligosaccharides (RFOs) are involved in the storage and transport of carbon and serve as compatible solutes for protection against abiotic stresses like drought or cold. RFOs are usually transported in plant species that load sugars symplastically into the phloem. Loading probably occurs by a polymer trapping mechanism which establishes a concentration gradient of assimilates between the mesophyll and the vasculature. Transgenic approaches have demonstrated phloem transport of small molecules produced in the companion cells of apoplastic loading species, but these molecules have been non-native transport substances to plants. In this study, transgenic potato plants with constitutive or companion cell specific overexpression of galactinol synthase (GS) or GS plus raffinose synthase (RS) are characterized, which together provide new insights into the metabolism and transport of RFOs in plants. It is demonstrated that raffinose and galactinol are both transported in the phloem and that, whilst the effect of GS overexpression is promoter-independent, that of RS is dependent on the promoter used. The presence of significant amounts of galactinol in the phloem is shown and also that transgenic potato is unable to transport large amounts of raffinose despite high RS expression and substrate concentrations. These data indicate that there may be additional features of intermediary cells, the specialized companion cells of RFO transporting plants, required for significant RFO synthesis and transport that are currently not well-understood.     

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine     

Inorganic nitrogen assimilation in the non-N2-fixing cyanobacterium Phormidium laminosum. I. Cellular levels of glutamine synthetase and NADPH-dependent glutamate dehydrogenase

Cell-free extracts of nitrate-grown as well as of ammonium-grown cells of the filamentous non-nitrogen-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) showed detectable levels of both glutamine synthetase (GS, EC 6.3.1.2) and NADPH-dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) activities. The GS level of nitrate-grown cells was higher than that of ammonium-grown cells, whereas the GDH level was higher in ammonium-grown cells and depended on the external ammonium concentration. When nitrate-grown cells were transferred to an ammonium-containing medium, a decrease of GS and an increase of GDH specific activities occurred, even in the presence of nitrate. Conversely, when ammonia-grown cells were transferred to a nitrate-containing medium, an increase of GS and a decrease of GDH-specific activities took place. Both these effects were inhibited by chloramphenicol and were probably mediated by de novo protein synthesis. When either cell type was transferred to a medium without nitrogen source, the specific activities of both enzymes increased. When nitrate-grown cells were transferred to nitrate medium with L-methionine-DL-sulphoximine (MSX) added, the specific activity of GDH also increased. Here we present some evidence that, under certain conditions of nitrogen availability, GDH would play a minor role in ammonium assimilation.     

The two senescence-related markers, GS1 (cytosolic glutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in Nicotiana tabacum L. leaves

To investigate the role of stress in nitrogen management in plants, the effect of pathogen attack, elicitors, and phytohormone application on the expression of the two senescence-related markers GS1 (cytosolic glutamine synthetase EC 6.3.1.2) and GDH (glutamate dehydrogenase, EC 1.4.1.2) involved in nitrogen mobilization in senescing leaves of tobacco (Nicotiana tabacum L.) plants, was studied. The expression of genes involved in primary nitrogen assimilation such as GS2 (chloroplastic glutamine synthetase) and Nia (nitrate reductase, EC 1.6.1.1) was also analysed. The Glubas gene, coding a beta-1,3-glucanase, was used as a plant-defence gene control. As during natural senescence, the expression of GS2 and Nia was repressed under almost all stress conditions. By contrast, GS1 and GDH mRNA accumulation was increased. However, GS1 and GDH showed differential patterns of expression depending on the stress applied. The expression of GS1 appeared more selective than GDH. Results indicate that the GDH and GS1 genes involved in leaf senescence are also a component of the plant defence response during plant-pathogen interaction. The links between natural plant senescence and stress-induced senescence are discussed, as well as the potential role of GS1 and GDH in a metabolic safeguard process.     

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     

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     

Glutamine synthetase and glutamate dehydrogenase isoforms in maize leaves: localization, relative proportion and their role in ammonium assimilation or nitrogen transport

 Mesophyll cells (MCs) and bundle-sheath cells (BSCs) of leaves of the C₄ plant maize (Zea mays L.) were separated by cellulase digestion to determine the relative proportion of the glutamine synthetase (GS; EC 6.3.1.2) or the NADH-glutamate dehydrogenase (GDH; EC 1.4.1.2) isoforms in each cell type. The degree of cross-contamination between our MC and BSC preparations was checked by the analysis of marker proteins in each fraction. Nitrate reductase (EC 1.6.6.1) proteins (110 kDa) were found only in the MC fraction. In contrast, ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) proteins (160 kDa) were almost exclusively present in the BSC fraction. These results are consistent with the known intercellular distribution of nitrate reductase and Fd-GOGAT proteins in maize leaves and show that the cross-contamination between our MC and BSC fractions was very low. Proteins corresponding to cytosolic GS (GS-1) or plastidic GS (GS-2) were found in both the MC and BSC fractions. While equal levels of GS-1 (40 kDa) and GS-2 (44 kDa) polypeptides were present in the BSC fraction, the GS-1 protein level in the MC fraction was 1.8-fold higher than the GS-2 protein pool. Following separation of the GS isoforms by anion-exchange chromatography of MC or BSC soluble protein extracts, the relative GS-1 activity in the MC fraction was found to be higher than the relative GS-2 activity. In the BSC fraction, the relative GS-1 activity was very similar to the relative GS-2 activity. Two isoforms of GDH with apparent molecular weights of 41 kDa and 42 kDa, respectively, were detected in the BSC fraction of maize leaves. Both GDH isoenzymes appear to be absent from the MC fraction. In the BSCs, the level of the 42-kDa GDH isoform was 1.7-fold higher than the level of the 41-kDa GDH isoform. A possible role for GS-1 and GDH co-acting in the synthesis of glutamine for the transport of nitrogen is discussed. Received: 25 January 2000 / Accepted: 30 March 2000     

Enzymes of Ammonia Assimilation, Photosynthesis, and Respiration in Alfalfa Leaves of Different Ages

The activities of enzymes involved in ammonia metabolism ferredoxin-dependent glutamate synthase (Fd-GOGAT), glutamine synthetase (GS) and glutamate dehydrogenase (GDH), the rates of photosynthetic oxygen evolution, dark respiration, and the activity of RuBP carboxylase (RuBPC) were determined in alfalfa (Medicago sativa L.) leaves taken from the apex (apical leaves), from the second to the fourth internode (mature leaves) and from the bottom of the canopy (basal leaves). Photosynthetic rate and the activities of RuBPC, GS and Fd-GOGAT showed their maximum in the mature leaves. The respiration rate together with amino acid and ammonium contents decreased with leaf age, whereas the opposite was true for GDH activity. Basal leaves still maintained substantial levels of chlorophylls, GS and Fd-GOGAT activities and oxygen evolution rate, thus suggesting that photosynthesis has some role in the reassimilation of the nitrogen liberated during protein degradation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

Bottle gourd rootstock-grafting affects nitrogen metabolism in NaCl-stressed watermelon leaves and enhances short-term salt tolerance

The plant growth, nitrogen absorption, and assimilation in watermelon (Citrullus lanatus [Thunb.] Mansf.) were investigated in self-grafted and grafted seedlings using the salt-tolerant bottle gourd rootstock Chaofeng Kangshengwang (Lagenaria siceraria Standl.) exposed to 100 mM NaCl for 3 d. The biomass and NO₃⁻ uptake rate were significantly increased by rootstock while these values were remarkably decreased by salt stress. However, compared with self-grafted plants, rootstock-grafted plants showed higher salt tolerance with higher biomass and NO₃⁻ uptake rate under salt stress. Salinity induced strong accumulation of nitrate, ammonium and protein contents and a significant decrease of nitrogen content and the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) in leaves of self-grafted seedlings. In contrast, salt stress caused a remarkable decrease in nitrate content and the activities of GS and GOGAT, and a significant increase of ammonium, protein, and nitrogen contents and NR activity, in leaves of rootstock-grafted seedlings. Compared with that of self-grafted seedlings, the ammonium content in leaves of rootstock-grafted seedlings was much lower under salt stress. Glutamate dehydrogenase (GDH) activity was notably enhanced in leaves of rootstock-grafted seedlings, whereas it was significantly inhibited in leaves of self-grafted seedlings, under salinity stress. Three GDH isozymes were isolated by native gel electrophoresis and their expressions were greatly enhanced in leaves of rootstock-grafted seedlings than those of self-grafted seedlings under both normal and salt-stress conditions. These results indicated that the salt tolerance of rootstock-grafted seedlings might (be enhanced) owing to the higher nitrogen absorption and the higher activities of enzymes for nitrogen assimilation induced by the rootstock. Furthermore, the detoxification of ammonium by GDH when the GS/GOGAT pathway was inhibited under salt stress might play an important role in the release of salt stress in rootstock-grafted seedlings.     

氮素水平对花生氮素代谢及相关酶活性的影响

 在大田高产条件下研究了氮素水平对花生(Arachis hypogaea)可溶性蛋白质、游离氨基酸含量及氮代谢相关酶活性的影响, 结果表明, 适当提高氮素水平既能增加花生各器官中可溶性蛋白质和游离氨基酸的含量, 又能提高硝酸还原酶、谷氨酰胺合成酶和谷氨酸脱氢酶等氮素同化酶的活性, 使其达到同步增加; 氮素水平过高虽能提高硝酸还原酶和籽仁蛋白质含量, 但谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH)的活性下降; N素施肥水平不改变花生植株各器官中可溶性蛋白质、游离氨基酸含量以及硝酸还原酶(NR)、谷氨酰胺合成酶、谷氨酸脱氢酶活性的变化趋势, 但适量施N (A2和A3处理)使花生各营养器官中GS、GDH活性提高; 氮素水平对花生各叶片和籽仁中GS、GDH活性的高低影响较大, 但对茎和根中GDH活性大小的影响较小。