Assimilation of Ammonium Nitrogen by Heterotrophic and Symbiotrophic White Lupine Plants

The activities of glutamate dehydrogenase, asparagine synthetase, and total glutamine synthetase in the organs of the white lupine (Lupinus albus L.) plants were measured during plant growth and development. In addition, the dynamics of free amino acids and amides in plant organs was followed. It was shown that the change in the nutrition type was important for controlling enzyme activities in the organs examined and, consequently, for directing the pathway of ammonium nitrogen assimilation. As long as the plants remained heterotrophic, glutamine-dependent asparagine synthetase of cotyledons and glutamine synthetase of leaves apparently played a major role in the assimilation of ammonium nitrogen. In symbiotrophic plants, root nodules became an exclusive site of asparagine synthesis, and the role of leaf glutamine synthetase increased. Unlike glutamine synthetase and asparagine synthetase, glutamate dehydrogenase activity was present in all organs examined and was less dependent on the nutrition type. This was also indicated by a weak correlation of glutamate dehydrogenase activity with the dynamics of free amino acid and amide content in these organs. It is supposed that glutamine synthetase plays a leading role in both the primary assimilation of ammonium, produced during symbiotic fixation of molecular nitrogen in root nodules, and in its secondary assimilation in cotyledons and leaves. On the other hand, secondary nitrogen assimilation in the axial organs occurs via an alternative glutamate dehydrogenase pathway.     

Amino acid metabolism associated with N-mobilization from the flag leaf of wheat (Triticum aestivum L.) during grain development

Field-grown winter wheat (Triticum aestivum L. cv. Castell) was used to study changes in the free amino acid pools of different plant parts and related enzyme activities in the flag leaf throughout the grain-filling period in three consecutive growing seasons. Amino acid analysis data indicated that, during senescence, the nitrogen flow in the flag leaf was directed towards the synthesis of glutamine as a specific nitrogen transport form. Of the enzymes involved, total glutamine synthetase (GS; EC 6.3.1.2) and especially ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) activities declined continuously as senescence progressed. Unlike (chloroplastic) GS₂, (cytosolic) GS₁ was shown to be very persistent suggesting a special role for this isoenzyme in the N-reallocation process. Glutamate-oxaloacetate transaminase (GOT; EC 2.6.1.1), glutamate-pyruvate transaminase (GPT; EC 2.6.1.2) and isocitrate dehydrogenase (IDH; EC 1.1.1.42) showed a characteristic biphasic activity profile after anthesis. It is proposed that these enzymes, for each of which at least two isoenzymes were demonstrated, are involved in glutamate synthesis at the later stages of leaf senescence. Ammonium levels were fairly constant throughout the flag leafs life span, an ultimate rise often following peak values of glutamate dehydrogenase (GDH; EC 1.4.1.4) activity. The enzymology of flag leaf amino acid metabolism during grain development is further discussed in relation to observations of NH₃-volatilization from naturally senescing wheat plants.     

Enzymology of Glutamine Metabolism Related to Senescence and Seed Development in the Pea (Pisum sativum L.)

The metabolism of glutamine in the leaf and subtended fruit of the aging pea (Pisum sativum L. cv. Burpeeana) has been studied in relation to changes in the protein, chlorophyll, and free amino acid content of each organ during ontogenesis. Glutamine synthetase [EC 6.3.1.2] activity was measured during development and senescence in each organ. Glutamate synthetase [EC 2.6.1.53] activity was followed in the pod and cotyledon during development and maturation. Maximal glutamine synthetase activity and free amino acid accumulation occurred together in the young leaf. Glutamine synthetase (in vitro) in leaf extracts greatly exceeded the requirement (in vivo) for reduced N in the organ. Glutamine synthetase activity, although declining in the senescing leaf, was sufficient (in vitro) to produce glutamine from all of the N released during protein hydrolysis (in vivo). Maximal glutamine synthetase activity in the pod was recorded 6 days after the peak accumulation of the free amino acids in this organ.

In the young pod, free amino acids accumulated as glutamate synthetase activity increased. Maximal pod glutamate synthetase activity occurred simultaneously with maximal leaf glutamine synthetase activity, but 6 days prior to the corresponding maximum of glutamine synthetase in the pod. Cotyledonary glutamate synthetase activity increased during the assimilatory phase of embryo growth which coincided with the loss of protein and free amino acids from the leaf and pod; maximal activity was recorded simultaneously with maximal pod glutamine synthetase.

We suggest that the activity of glutamine synthetase in the supply organs (leaf, pod) furnishes the translocated amide necessary for the N nutrition of the cotyledon. The subsequent activity of glutamate synthetase could provide a mechanism for the transfer of imported amide N to alpha amino N subsequently used in protein synthesis. In vitro measurements of enzyme activity indicate there was sufficient catalytic potential in vivo to accomplish these proposed roles.

     

Regulation of the ammonia assimilatory enzymes in Salmonella typhimurium.

The regulation of glutamate dehydrogenase (EC 1.4.1.4), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 2.6.1.53) was examined for cultures of Salmonella typhimurium grown with various nitrogen and amino acid sources. In contrast to the regulatory pattern observed in Klebsiella aerogenes, the glutamate dehydrogenase levels of S. typhimurium do not decrease when glutamine synthetase is derepressed during growth with limiting ammonia. Thus, it appears that the S. typhimurium glutamine synthetase does not regulate the synthesis of glutamate dehydrogenase as reported for K. aerogenes. The glutamate dehydrogenase activity does increase, however, during growth of a glutamate auxotroph with glutamate as a limiting amino acid source. The regulation of glutamate synthase levels is complex with the enzyme activity decreasing during growth with glutamate as a nitrogen source, and during growth of auxotrophs with either glutamine or glutamate as limiting amino acids.     

Enzymes of glutamine metabolism in testa-pericarp and endosperm of developing wheat grain

Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate puruvate transaminase and glutamate oxaloacetate transaminase have been assayed in developing testa-pericarp and endosperm of two wheat varieties, namely Shera (11.6% protein) and C-306 (9.8% protein). On per organ basis, activities of all the enzymes studied, except glutamine synthetase, increased during development. Glutamine synthetase activity decreased during development in the testa-pericarp, whereas, no glutamine synthetase activity could be detected in endosperm of either variety at any stage of development. Compared to testa-pericarp, endosperm had higher activities of glutamate synthase and glutamate pyruvate transaminase. On the whole, enzyme activities in Shera were higher, as compared to C-306. Developmental patterns and relative levels of enzyme activities in the two varieties were more or less the same, when expressed on dry weight basis or as specific activities. The results suggest that ammonia assimilation in developing wheat grain takes place by the glutamate dehydrogenase pathway in the endosperm; and both by the glutamate dehydrogenase and glutamine synthetase—glutamate synthase pathways in the testa-pericarp.     

The Role of Ammonium Assimilating Enzymes in Lentil Roots and Nodules

Activities of ammonium assimilating enzymes glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) as well as the amino acid content were higher in nodules compared to roots. Their activities increased at 40 and 60 d after sowing, with a peak at 90 d, a time of maximum nitrogenase activity. The GS/GOGAT ratio had a positive correlation with the amino acid content in nodules. Higher activities of AST than ALT may be due to lower glutamine and higher asparagine content in xylem. The data indicated that glutamine synthetase and glutamate synthase function as the main route for the assimilation of fixed N, while NADH-dependent glutamate dehydrogenase may function at higher NH₄ ⁺ concentration in young and senescing nodules. Enzyme activities in lentil roots reflected a capacity to assimilate N for making the amino acids they may need for both growth and export to upper parts of the plant. This revised version was published online in July 2006 with corrections to the Cover Date.     

Changes in the activities of enzymes involved in amino acid metabolism during the senescence of detached wheat leaves

The activities of several enzymes related to amino acid metabolism were investigated in senescing detached wheat leaves ( Triticum aestivum L. cv. Diplomat) in light and darkness and after kinetin treatment. Glutamine synthetase and glutamate synthase activities rapidly declined in darkness. In light, the decline of glutamate synthase activity was retarded, while the activity of glutamine synthetase remained high and even increased transitorily. Kinetin treatment counteracted the decline of the activities of both enzymes. The activity of glutamate dehydrogenase markedly increased during senescence, particularly in light, and kinetin treatment lowered its activity. The activities of glutamate-oxaloacetate and glutamate-pyruvate amino-transferases and of NADP-dependent isocitrate dehydrogenase also increased in detached wheat leaves in light. Kinetin treatment prevented the rise of these enzyme activities. In darkness, the activities of glutamate-oxaloacetate aminotransferase and NADP-dependent isocitrate dehydrogenase decreased slowly while the decline of glutamate-pyruvate aminotransferase activity was more rapid. The activity of NAD-dependent malate dehydrogenase decreased both in light and, more rapidly, in darkness. The pattern of changes of the enzyme activities provides an explanation for the amino acid transformations and the flow of amino nitrogen into transport metabolites in senescing leaves.     

Investigation of the site of synthesis of chIcoroplastic enzymes of nitrogen metabolism by the use of heat-treated 70S ribosomedeficient rye leaves

The activities of the enzymes nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (GOGAT; EC 1.4.7.1), glutamate-oxaloacetate aminotransferase (EC 2.6.1.1), and glutamate dehydrogenase (EC 1.4.1.2) were compared in light-grown green or etiolated leaves of rye seedlings ( Secale cereale L. cv. Halo) raised at 22°C, and in the bleached 70S ribosome-deficient leaves of rye seedlings grown at a non-permissive high temperature of 32°C. Under normal permissive growth conditions the activities of most of the enzymes were higher in light-grown, than in dark-grown, leaves. All enzyme activities assayed were also observed in the heat-treated 70S ribosome-deficient leaves. Glutamine synthetase, glutamate synthase, and glutamate-oxaloacetate aminotransferase occurred in purified ribosome-deficient plastids separated on sucrose gradients. For glutamate-oxaloacetate aminotransferase four multiple forms were separated by polyacrylamide gel electrophoresis from leaf extracts. The chloroplastic form of this enzyme was also present in 70S ribosome-deficient leaves. It is concluded that the chloroplast-localized enzymes nitrite reductase, glutamine synthetase, glutamate synthase and glutamate-oxaloacetate aminotransferase, or their chloroplast-specific isoenzyme forms, are synthesized on cytoplasmic 80S ribosomes.     

Qualitative and quantitative changes in nitrogenous compounds in senescing leaf and bark tissues of the apple

Quantitative and qualitative changes in proteins and ethanol-soluble nitrogen were followed in senescing leaf and bark tissues of ‘Golden Delicious’ apple trees (Malus domestica Borkh.). While senescing leaves lost 46% of their proteins, total bark protein increased 240% during senescence. However, the protein nitrogen gain in bark tissue was about the same as the protein nitrogen loss in leaf tissue per unit fresh weight of tissues. The pattern of bark protein accumulation appears to be gradual from early August to November and sequential from lower to higher molecular weight species of proteins. The final electrophoretic profile of total bark proteins was established at the later stages of senescence. By late November, 89% of the nitrogen in the bark tissue was found in proteins with 11% in the ethanol-soluble fractions. The total protein content of dormant bark tissue was 3.5% per gram dry tissue. Fractionation of the total bark proteins by DEAE-cellulose chromatography indicated that the final upsurge of bark proteins observed in November was associated primarily with one group of proteins (Peak III).     

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The technique of EDTA-enhanced phloem exudation (King and Zeevaart, 1974: Plant Physiol. 53, 96–103) was evaluated with respect to the collection and identification of amino acids exported from senescing wheat leaves. Whilst the characteristics of the exudate collected conform with many of the accepted properties of phloem exudate, unexpectedly high molar proportions of phenylalanine and tyrosine were observed. By comparing exudation into a range chelator solutions with exudation into water, the increased exudation of phenylalanine and tyrosine relative to the other amino acids occurring when ethylene-diaminetetracetic acid was used, was considered to an artefact.In plants thought to be relying heavily on mobilisation of protein reserves to satisfy the nitrogen requirements of the grain, the major amino acids present in flag-leaf phloem exudate were glutamate, aspartate, serine, alanine and glycine. Only small proportions of amides were present until late in senescence when glutamine became the major amino acid in phloem exudate (25 molar-%). Glutamine was always the major amino acid in xylem sap (50 molar-%).The activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), glutamate dehydrogenase (EC 1.4.1.3) and asparagine synthetase (EC 5.3.5.4) were measured in the flag leaf throughout the grain-filling period. Glutamine synthetase and glutamate-synthase activities declined during this period. Glutamate-dehydrogenase activity was markedly unchanged despite variation in the number of multiple forms visualised after gel electrophoresis. The activity of the enzyme reached a peak only very late in the course of senescence of the flag leaf. No asparagine-synthetase activity could be detected in the flag leaf during the grain-filling period.II. Peoples et al. (1980)     

The localisation of enzymes of nitrogen assimilation in maize leaves and their activities during greening

The activities of nitrate reductase (EC1.6.6.1), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC6.3.1.2), glutamate synthase (EC1.4.7.1) and NAD(P)H-dependent glutamate dehydrogenase (EC 1.4.1.3) were investigated in mesophyll and bundle sheath cells of maize leaves (Zea mays L.). Whereas nitrate and nitrite reductase appear to be restricted to the mesophyll and GDH to the bundle sheath, glutamine synthetase and glutamate synthase are active in both tissues.During the greening process, the activities of nitrate and nitrite reductase increased markedly, but glutamine synthetase, glutamate synthase and glutamate dehydrogenase changed little.Abbreviations BDH British Drug Houses - EDTA Ethylene diamine tetra-acetic acid - GDH Glutamate dehydrogenase - NADH Nicotinamide-adenine dinucleotide reduced form - NADPH Nicotnamide-adenine dinucleotide phosphate reduced form - PMSF Phenylmethyl sulphonyl fluoride     

Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.

The two isoenzymes of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), previously identified in root nodules of Phaseolus vulgaris L., have both been shown to be located in root-nodule plastids. The nodule specific NADH-GOGAT II accounts for the majority of the activity in root nodules, and is present almost exclusively in the central tissue of the nodule. However about 20% of NADH-GOGAT I activity is present in the nodule cortex, at about the same specific activity as this isoenzyme is found in the central tissue. Glutamine synthetase (GS; EC 6.3.1.2) occurs predominantly as the polypeptide in the central tissue, whereas in the cortex, the enzyme is represented mainly by the polypeptide. Over 90% of both GS and NADH-GOGAT activities are located in the central tissue of the nodule and GS activity exceeds NADH-GOGAT activity by about twofold in this region. Using the above information, a model for the subcellular location and stoichiometry of nitrogen metabolism in the central tissue of P. vulgaris root nodules is presented.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GOGAT glutamate synthase - GS glutamine synthetase - NADH-GOGAT NADH-dependent glutamate synthase - IEX-HPLC ion-exchange high-performance liquid chromatography     

The pathways of ammonium assimilation in Rhizobium meliloti

Two pathways of ammonium assimilation are known in bacteria, one mediated by glutamate dehydrogenase, the other by glutamine synthetase and glutamate synthase. The activities of these three enzymes were measured in crude extracts from four Rhizobium meliloti wild-type strains, 2011, M15S, 444 and 12. All the strains had active glutamine synthetase and NADP-linked glutamate synthase. Assimilatory glutamate dehydrogenase activity was present in strains 2011, M15S, 444, but not in strain 12. Three glutamate synthase deficient mutants were isolated from strain 2011. They were unable to use 1 mM ammonium as a sole nitrogen source. However, increased ammonium concentration allowed these mutants to assimilate ammonium via glutamate dehydrogenase. It was found that the sole mode of ammonium assimilation in strain 12 is the glutamine synthetase-glutamate synthase route; whereas the two pathways are functional in strain 2011.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase     

Intracellular distribution of enzymes associated with nitrogen assimilation in roots

The cellular distribution of enzymes involved in nitrogen assimilation: nitrate reductase (EC 1.6.6.2), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) has been studied in the roots of five plants: maize (Zea mays L. hybrid W 64A x W 182E), rice (Oryza sativa L. cv. Delta), bean (Phaseolus vulgaris L. cv. Contender), pea (Pisum sativum L. cv. Demi-nain), and barley (Hordeum vulgare L.). Initially, cell organelles were separated from soluble proteins by differential centrifugation. Cell organelles were also subjected to sucrose density gradients. The results obtained by these two methods indicate that nitrite reductase and glutamate synthase are localized in plastids, nitrate reductase and glutamine synthetase are present in the cytosol, and glutamate dehydrogenase is a mitochondrial enzyme.     

Ligninolytic activity and levels of ammonia assimilating enzymes in Sporotrichum pulverulentum

Levels of ammonia-assimilating enzymes (glutamate dehydrogenase, glutamine synthetase, glutamate synthase) were determined in extracts of Sporotrichum pulverulentum grown under different conditions with respect to both nitrogen source and concentration. Evolution of ¹⁴CO₂ from ¹⁴C-synthetic lignin by fungal cultures grown under parallel conditions was also determined as a measure of lignin decomposition and the suppressive effect of nitrogen on ligninolysis confirmed. Under low nitrogen conditions, fungal extracts exhibited relatively high levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase dehydrogenase. Conversely, in high nitrogen extracts, lower levels of NADP-dependent glutamate dehydrogenase and glutamine synthetase activity, and higher levels of NAD-dependent glutamate dehydrogenase, were recorded. Possible effects of enzyme activities on intracellular pool concentrations of glutamate/glutamine, and the implications for the regulation of lignin metabolism, are discussed.A preliminary report was presented at The Ekman Days 1981, International Symposium on Wood and Pulping Chemistry, Stockholm, Sweden, June 9–12, 1981.     

Reinvestigation of the proposed red light effect on carotenogenesis in the fungus Verticillium agaricinum

The activities of glutamine synthetase (EC 6.3.1.2) and glutamate dehydrogenase (EC 1.4.1.2) appear to be inversely related in their distribution among the different tissues of 40-day-old tomato plants ( Lycopersicon esculentum L. cv. Hellfrucht Frühstamm), glutamine synthetase activity being highest in the leaves and glutamate dehydrogenase activity in the root. Leaf glutamine synthetase activity decreases with plant growth and shows diurnal variation with a maximum in the light and a minimum in the dark. In vitro, the activity of purified glutamine synthetase increases with the energy charge of the assay medium and decreases with increasing concentrations of p -chloromercuribenzoic acid. Glutamine synthetase activity in the plant may be regulated by physiological changes occurring during the light-dark transition periods.     

Nitrogen assimilation in facultative methylotrophic bacteria

A study was done of the pathways of nitrogen assimilation in the facultative methylotrophsPseudomonas MA andPseudomonas AM1, with ammonia or methylamine as nitrogen sources and with methylamine or succinate as carbon sources. When methylamine was the sole carbon and/or nitrogen source, both organisms possessed enzymes of the glutamine synthetase/glutamate synthase pathway, but when ammonia was the nitrogen sourcePseudomonas AM1 also synthesized glutamate dehydrogenase with a pH optimum of 9.0, andPseudomonas MA elaborated both glutamate dehydrogenase (pH optimum 7.5) and alanine dehydrogenase (pH optimum 9.0). Glutamate dehydrogenase and glutamate synthase from both organisms were solely NADPH-dependent; alanine dehydrogenase was NADH-dependent. No evidence was obtained for regulation of glutamine synthetase by adenylylation in either organism, nor did glutamine synthetase appear to regulate glutamate dehydrogenase synthesis.     

The pathway of nitrogen assimilation in plants

The major route of nitrogen assimilation has been considered for many years to occur via the reductive amination of α-oxoglutarate, catalysed by glutamate dehydrogenase. However, recent work has shown that in most bacteria an alternative route via glutamine synthetase and glutamine: 2-oxoglutarate aminotransferase (glutamate synthase) operates under conditions of ammonia limitation. Subsequently the presence of a ferredoxin-dependent glutamate synthase in green leaves and green and blue-green algae, and a NAD(P)H and ferredoxin-dependent enzyme in roots and other non-green plant tissues, has suggested that this route may also function in most members of the plant kingdom. The only exceptions are probably the majority of the fungi, where so far most organisms studied do not appear to contain glutamate synthase. Besides the presence of the necessary enzymes there is other evidence to support the contention that the assimilation of ammonia into amino acids occurs via glutamine synthetase and glutamate synthase, and that it is unlikely that glutamate dehydrogenase plays a major role in nitrogen assimilation in bacteria or higher plants except in circumstances of ammonia excess.     

Changes in glutamate dehydrogenase activity of Chlamydomonas reinhardtii under different trophic and stress conditions

Abstract. Under stress conditions (darkness, nitrogen starvation, high ammonium concentrations, glutamine synthetase and glutamate synthase inhibition) glutamate dehydrogenase animating activity levels of Chlamydomonas cells varied inversely to those of glutamine synthetase. Nitrogen and carbon sources also influenced glutamate dehydrogenase levels in Chlamydomonas , the highest values being found in cells cultured mixotrophically with ammonium, under which conditions glutamate dehydrogenase and glutamine synthetase levels were likewise inversely related. These facts, together with the analysis of internal fluctuations of ammonium, 2-oxoglutarate, and the amino acid pool as well as the variations of certain enzymes involved in carbon metabolism indicate that glutamate dehydrogenase animating activity is adaptative, being involved in the maintenance of intracellular levels of L-glutamate when they cannot be maintained by the GS-GOGAT cycle, and probably more connected with carbon than nitrogen metabolism.     

Nitrogen assimilation in opaque and normal sorghum during grain development

Activity of key nitrogen assimilating enzymes was studied in developing grains of high-lysine opaque sorghum P-721 and normal sorghum CSV-5. The higher percentage of protein in opaque sorghum was mainly due to lower starch content since protein per grain was less than in CSV-5. During grain development, albufn and globulin decreased while prolafne and glutelin increased. Prolafne content in CSV-5 was higher than in opaque sorghum. Average nitrate reductase activity in flag and long leaf were similar in both the varieties. The nitrate reductase activity decreased during grain development. Glutamate dehydrogenase activity was higher during early development and lower at later stages in opaque sorghum than in CSV-5. Glutamate oxaloacetate transaminase activity was higher and glutamine synthetase lower in opaque sorghum than in CSV-5 grains during development. Glutamate synthase activity was higher in opaque sorghum up to day 20 and lower thereafter than in CSV-5. It is suggested that reduced activities of glutamine synthetase as well as glutamate synthase in opaque sorghum as compared to CSV-5 during later stages of development may restrict protein accumulation in the former.