Ammonium Assimilation

Chloroplast located Glutamine Synthetase (GS2) is believed to play a major role in the reassimilation of ammonium generated by photorespiration, being GS2 knockout mutants unable to grow under photorespiratory conditions (low-CO 2 atmosphere) in the species characterized so far (Barley, Lotus). To investigate the importance of GS2 in A. thaliana nitrogen metabolism mutant plants devoid of this GS isoenzyme were characterized. It was shown that GS2 mutants although smaller, slightly chlorotic and with the nitrogen metabolism impaired, were able to grow and complete their life cycle under ordinary air conditions. Surprisingly, GS2 mutants were more tolerant to salt stress than wild-type plants. The lack of GS2 seems to be compensated by higher expression of some GS cytosolic isogenes, namely GLN1;2 and GLN1;3 and by glutamate dehydrogenase, whose activity and expression is enhanced in the GS2 mutant plants and might account for the increased tolerance to salt stress. Under conditions that minimize photorespiration (CO2-enriched atmosphere) plant growth and ammonium assimilation impairment is less evident in the GS2 mutant plants and is accompanied by an adjustment of levels of expression of the cytosolic isogenes, with an increase in the expression of GLN1;3 and a decrease in the expression of the GLN1;1 and GLN1;2. Altogether the results confirm a major role of GS2 in the assimilation of ammonium released during photorespiration, but suggest a redundancy of activity with cytosolic GSs and GDH and further support the involvement of the chloroplastic isoenzyme in primary nitrogen assimilation and plant growth and development in A. thaliana.

Sulfate-reducing bacteria (SRB) are a diverse group of anaerobic microorganisms that obtain their energy from dissimilatory sulfate reduction. Some SRB species have high respiratory versatility due to the possible use of alternative electron acceptors. A good example is Desulfovibrio desulfuricans ATCC 27774, which grows in the presence of nitrate (end product: ammonium) with higher rates and yields to those observed in sulfate containing medium (end product: sulfide). In this work, the mechanisms supporting the respiratory versatility of D. desulfuricans were unraveled through the analysis of the proteome of the bacterium under different experimental conditions. The most remarkable difference in the two-dimensional gel electrophoresis maps is the high number of spots exclusively represented in the nitrate medium. Most of the proteins with increase abundance are involved in the energy metabolism and the biosynthesis of amino acids (or proteins), especially those participating in ammonium assimilation processes. qPCR analysis performed during different stages of the bacterium's growth showed that the genes involved in nitrate and nitrite reduction (napA and nrfA, respectively) have different expressions profiles: while napA did not vary significantly, nrfA was highly expressed at a 6 h time point. Nitrite levels measured along the growth curve revealed a peak at 3 h. Thus, the initial consumption of nitrate and concomitant production of nitrite must induce nrfA expression. The activation of alternative mechanisms for energy production, aside several N-assimilation metabolisms and detoxification processes, solves potential survival problems in adapting to different environments and contributes to higher bacterial growth rates.

MATERIALS AND METHODS In the seedcoats of developing pea seeds, the maximal activities of asparaginase (EC 3.5.1.1) and aspartate: a-ketoglutarate aminotransferase (EC 2.6.1.1) are attained early in development, before the embryo has expanded to fill the embryo sac. These two enzyme activities could account for the early absence of asparagine and aspartate from the fluid secreted by the seedcoats into the embryo sac. Changes in the activities of alanine: a-ketoglutarate aminotransferase (EC 2.6.1.2), glutamate dehydrogenase (EC 1.4.1.3), glutamine synthetase

Ammonium assimilation is linked to fundamental cellular processes that include the synthesis of non-essential amino acids like glutamate and glutamine. In Saccharomyces cerevisiae glutamate can be synthesized from α-ketoglutarate and ammonium through the action of NADP-dependent glutamate dehydrogenases Gdh1 and Gdh3. Gdh1 and Gdh3 are evolutionarily adapted isoforms and cover the anabolic role of the GDH-pathway. Here, we review the role and function of the GDH pathway in glutamate metabolism and we discuss the additional contributions of the pathway in chromatin regulation, nitrogen catabolite repression, ROS-mediated apoptosis, iron deficiency and sphingolipid-dependent actin cytoskeleton modulation in S.cerevisiae. The pleiotropic effects of GDH pathway in yeast biology highlight the importance of glutamate homeostasis in vital cellular processes and reveal new features for conserved enzymes that were primarily characterized for their metabolic capacity. These newly described features constitute insights that can be utilized for challenges regarding genetic engineering of glutamate homeostasis and maintenance of redox balances, biosynthesis of important metabolites and production of organic substrates. We also conclude that the discussed pleiotropic features intersect with basic metabolism and set a new background for further glutamate-dependent applied research of biotechnological interest.

Ectomycorrhizas, the dominating mycorrhizal symbiosis in boreal, temperate and some tropical forests, are formed by 5000^6000 species of the asco-and basidiomycetes. This high diversity of fungal partners allows optimal foraging and mobilisation of various nitrogen and phosphorus forms from organic soil layers. In this review, two approaches to study the functioning of this multitude of symbiotic associations are presented. On selected culture models, physiological and molecular investigations have shown that the supply of hexoses has a key function in controlling the plant^fungus interaction via partner-specific regulation of gene expression. Environmental factors which affect fungal carbon supply, such as increased nitrogen availability, also affect mycorrhiza formation. Based on such laboratory results, the adaptative capability of ectomycorrhizas to changing field conditions is discussed. The second approach consists of analysing the distribution of mycorrhizas in ecosystem compartments and to relate distribution patterns to variations of ecological factors. Recent advances in identification of fungal partners in ectomycorrhizas by analysing the internal transcribed spacer of ribosomal DNA are presented, which can help to resolve sampling problems in field studies. The limits of the laboratory and the field approaches are discussed. Despite some problems, this combined approach is the most promising. Direct investigation of gene expression, which has been introduced for soil bacteria, will be difficult in the case of mycorrhizal fungi which constitute organisms with functionally varying structures.

Glutamine synthetase is an essential enzyme in ammonium assimilation and glutamine biosynthesis. The Haloferax mediterranei genome has two other glnA-type genes (glnA2 and glnA3) in addition to the glutamine synthetase gene glnA. To determine whether the glnA2 and glnA3 genes can replace glnA in nitrogen metabolism, we generated deletion mutants of glnA. The glnA deletion mutants could not be generated in a medium without glutamine, and thus, glnA is an essential gene in H. mediterranei. The glnA deletion mutant was achieved by adding 40 mM glutamine to the selective medium. This conditional HM26-ΔglnA mutant was characterised with different approaches in the presence of distinct nitrogen sources and nitrogen starvation. Transcriptomic analysis was performed to compare the expression profiles of the strains HM26-ΔglnA and HM26 under different growth conditions. The glnA deletion did not affect the expression of glnA2, glnA3 and nitrogen assimilation genes under nitrogen starvation. Moreover, the results showed that glnA, glnA2 and glnA3 were not expressed under the same conditions. These results indicated that glnA is an essential gene for H. mediterranei and, therefore, glnA2 and glnA3 cannot replace glnA in the conditions analysed.

The nicotinamide adenine dinucleotide-specific glutamate dehydrogen- ase (L-glutamate:NAD' oxidoreductase, EC 1.4.1.2) of Chlorella soroki- niana was purified 1,000-fold to electrophoretic homogeneity. The native enzyme was shown to have a molecular weight of 180,000 and to be composed of four identical subunits with a molecular weight of 45,000. The N-terminal amino acid was determined to be lysine. The pH optima for the aminating and deaminating reactions were approximately 8 and 9, respec-

The ontogeny of glutamine synthetase (EC6.3.1.2), glutamate synthase (EC 1.4.1.14), asparagine synthetase (EC6.4.5.4) and glutamate dehydrogenase (EC 1.4.1.2) has been followed in cotyledons, roots, stems and leaves of young plants of Canavalia ensiformis. The four enzymes were found in all regions. Glutamine synthetase activity increased in the leaves with time and remained low in the cotyledons. Initially glutamate synthase activity in the cotyledons was NADH-dependent but later became Fd-dependent. Asparagine synthetase specific activity was highest in the roots until day 5 after which activity was similar in all regions. While glutamate dehydrogenase specific activity decreased with time in the leaves it increased in the cotyledons and roots. We suggest that in the early stages of seedling growth asparagine synthetase and glutamate dehydrogenase both play a fundamental role in the reassimilation of ammonium released from canavanine. When the plant acquires full photosynthetic capacity the glutamine synthetase/glutamate synthase pathway becomes the principal pathway of reassimilation in the aerial parts of the plant.

In higher plants it is now generally considered that glutamate dehydrogenase (GDH) plays only a small or negligible role in ammonia assimilation. To test this specific point, comparative studies of (15)NH(4) (+) assimilation were undertaken with a GDH1-null mutant of Zea mays and a related (but not strictly isogenic) GDH1-positive wild type from which this mutant was derived. The kinetics of (15)NH(4) (+) assimilation into free amino acids and total reduced nitrogen were monitored in both roots and shoots of 2-week-old seedlings supplied with 5 millimolar 99% ((15)NH(4))(2)SO(4) via the aerated root medium in hydroponic culture over a 24-h period. The GDH1-null mutant, with a 10- to 15-fold lower total root GDH activity in comparison to the wild type, was found to exhibit a 40 to 50% lower rate of (15)NH(4) (+) assimilation into total reduced nitrogen. Observed rates of root ammonium assimilation were 5.9 and 3.1 micromoles per hour per gram fresh weight for the wild type and mutant...

L-Alanine dehydrogenase was found in extracts of the antibiotic producer Streptornyces clavutigerus. The enzyme was induced by ammonia, and the leveI of induction was dependend on the extracellular concentratiom L-Alanine was the only amino acid able to induce atanine dehydrogenase. The enzyme was characterized from a 38-fold purified preparation. Pyruvate (K m = 1.1 raM), ammonia (K m = 20 mM) and NADH (K,, = 0.14 raM) were required for the reductive amination, and L-alanine (K,~ = 9.1 mM) and NAD (K,, = 0.5 raM) for the oxidative deaminating reaction. The aminating reaction was inhibited by alanine, serine and NADPH. Alanine inhibited uncompetitively with respect to NADH (K~ = 1.6 raM) and noncompetitively with respect to ammonia (K~ = 2.0 mM) and pyruvate (K~ = 3.0 mM). In the aminating reaction 3-hydroxypyruvate, glyoxylate and 2-oxobutyrate could partially (6 ~-7 ~/~) substitute pyruvate. Atanine dehydrogenase from & clavulige~us differed with respect to its molecular weight (92000) and its kinetic properties from those described for other microorganisms.

Respective roles of the glutamine synthetase/glutamate synthase cycle and glutamate dehydrogenase in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula Abstract Our objective was to determine the respective roles of the couple glutamine synthetase/glutamate synthase (GS/GOGAT) and glutamate dehydrogenase (GDH) in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula Gaertn. For this aim, amino acids were analyzed by HPLC and changes in gene expression of several enzymes involved in N and C metabolism were studied by real-time quantitative reverse transcription-polymerase chain reaction. Among the enzymes studied, GDH showed the highest increase in gene expression (80-fold), specifically in the embryo axis and concomitant with the increase in ammonium content during post-germinative growth. In cotyledons, GDH gene expression was very low. Although in vitro GDH aminating activity was several times higher than its deaminating activity, in vivo 15 NH 4 incorporation into amino acids was completely inhibited by methionine sulfoximine, a GS inhibitor, indicating that GDH is not involved in ammonium assimilation/detoxification. Changes in the expressions of GS and GOGAT isoforms revealed that GS1b (EC 6.3.1.2) in concert with NADH-dependent GOGAT (EC 1.4.1.14) constitute the major route of assimilation of ammonium derived from reserve mobilization and glutamic acid/glutamine syn-thesis in germinating M. truncatula seeds. However, during post-germinative growth, although germination was held in darkness, expression of GS2 and Fd-GOGAT (EC 1.4.7.1) increased and expression of GS1b decreased in cotyledons but not in the embryo axis. 2-Oxoglutarate, the substrate of the transamination reaction, was provided by the cytosolic isoform of isocitrate dehydrogenase (EC 1.1.1.42). We suggest that GDH during post-germinative growth, specifically in the developing embryo axis, contributes to ammonium delivery to GS for glutamine synthesis in the absence of primary NO 3 ) assimilation. Interestingly, this reaction also produces reducing power (NADH) in organs deprived of photosynthesis. Keywords Amino acid metabolism AE Germination AE Glutamate dehydrogenase AE Glutamine synthetase/ glutamate synthase AE Medicago AE Post-germinative growth Abbreviations EST: Expressed sequence tag AE Fd-GOGAT: Ferredoxin-dependent glutamate synthase AE GDH: Glutamate dehydrogenase AE GS: Glutamine synthetase AE GS1a, GS1b: Cytosolic GS AE GS2: Chloroplastic GS AE cICDH: Cytosolic isocitrate dehydrogenase AE mIDH: Mitochondrial isocitrate dehydrogenase AE MSC27: Medicago sativa cDNA 27 AE MSX: Methionine sulfoximine AE NADH-GOGAT: NADH-dependent glutamate synthase AE PEPc: Phosphoenolpyruvate carboxylase

Beggiatoa alba BI8LD was investigated for its pathways of ammonia assimilation. The increase in growth yields of B. alba with excess acetate was linear from 0.1 to 2.0 mM ammonia. B. alba had strong glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, irrespective of the ammonia concentration in the medium. Glutamate dehydrogenase activity was not found, and alanine dehydrogenase (aminating) was observed only when B. alba was grown at high (2.0 raM) ammonia. Methionine sulfoximine, an inhibitor of GS, inhibited growth of B. alba irrespective of the ammonia concentration in the medium. Thus it appears the primary pathway for ammonia assimilation in B. alba is via the GS-GOGAT pathway at both low and high ammonia concentrations. Preliminary experiments were unable to discern if the B. alba GS is modified by covalent modification.

Soluble amino acids in roots and primary amino acids, which were involved in primary ammonium assimilation, in the metabolites of 14 C-glucose fed to roots for 3 h in the dark were analyzed in the roots of non-nodulated soybean and pea plants grown in ammonium, nitrate or nitrogen-free media for 1 day. Compared with the effect of nitrate, ammonium supply strongly affected the content and synthesis of the amino acids in the roots. In both soybean and pea roots, the supply of ammonium increased considerably the concentrations of the primary amino acids, and asparagine was the most predominant amide, followed by glutamine. In nitrate-supplied soybean roots, the concentrations of asparagine, aspartate and alanine increased, but the concentration of glutamine was low. In the roots of pea plants grown in nitrate media, asparagine was the predominant amino acid, although the composition of the primary amino acids was little affected by nitrate supply. The proportion of amino acids synthesized from 14 C-glucose increased and asparagine rather than glutamine was predominantly synthesized in ammonium-supplied soybean and pea roots, whereas in nitrate-supplied roots asparagine was more actively synthesized than glutamine, although asparagine was not predominant. The ratio of C4 (asparagine + aspartate) to C5 (glutamine + glutamate) amino acids was twofold higher in ammonium-supplied and nitrate-supplied soybean roots than in roots receiving no nitrogen. In contrast, in pea roots, the C4/C5 ratio was twofold higher only in ammonium nutrition. The results obtained suggest that the roots of leguminous plants might possess an indigenous ability to provide a carbon skeleton for preferential synthesis of asparagine rather than glutamine with a high intensity of ammonium supply.

Respective roles of the glutamine synthetase/glutamate synthase cycle and glutamate dehydrogenase in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula Abstract Our objective was to determine the respective roles of the couple glutamine synthetase/glutamate synthase (GS/GOGAT) and glutamate dehydrogenase (GDH) in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula Gaertn. For this aim, amino acids were analyzed by HPLC and changes in gene expression of several enzymes involved in N and C metabolism were studied by real-time quantitative reverse transcription-polymerase chain reaction. Among the enzymes studied, GDH showed the highest increase in gene expression (80-fold), specifically in the embryo axis and concomitant with the increase in ammonium content during post-germinative growth. In cotyledons, GDH gene expression was very low. Although in vitro GDH aminating activity was several times higher than its deaminating activity, in vivo 15 NH 4 incorporation into amino acids was completely inhibited by methionine sulfoximine, a GS inhibitor, indicating that GDH is not involved in ammonium assimilation/detoxification. Changes in the expressions of GS and GOGAT isoforms revealed that GS1b (EC 6.3.1.2) in concert with NADH-dependent GOGAT (EC 1.4.1.14) constitute the major route of assimilation of ammonium derived from reserve mobilization and glutamic acid/glutamine syn-thesis in germinating M. truncatula seeds. However, during post-germinative growth, although germination was held in darkness, expression of GS2 and Fd-GOGAT (EC 1.4.7.1) increased and expression of GS1b decreased in cotyledons but not in the embryo axis. 2-Oxoglutarate, the substrate of the transamination reaction, was provided by the cytosolic isoform of isocitrate dehydrogenase (EC 1.1.1.42). We suggest that GDH during post-germinative growth, specifically in the developing embryo axis, contributes to ammonium delivery to GS for glutamine synthesis in the absence of primary NO 3 ) assimilation. Interestingly, this reaction also produces reducing power (NADH) in organs deprived of photosynthesis. Keywords Amino acid metabolism AE Germination AE Glutamate dehydrogenase AE Glutamine synthetase/ glutamate synthase AE Medicago AE Post-germinative growth Abbreviations EST: Expressed sequence tag AE Fd-GOGAT: Ferredoxin-dependent glutamate synthase AE GDH: Glutamate dehydrogenase AE GS: Glutamine synthetase AE GS1a, GS1b: Cytosolic GS AE GS2: Chloroplastic GS AE cICDH: Cytosolic isocitrate dehydrogenase AE mIDH: Mitochondrial isocitrate dehydrogenase AE MSC27: Medicago sativa cDNA 27 AE MSX: Methionine sulfoximine AE NADH-GOGAT: NADH-dependent glutamate synthase AE PEPc: Phosphoenolpyruvate carboxylase

During seed transition from quiescence to metabolic activity and subsequent germination, orthodox seeds become sensitive to stress. Aging seeds become particularly sensitive to stress conditions and during storage, seeds undergo aging that deteriorates their quality. Each seed lot contains strong seeds, weak seeds, and dead seeds, but dry seed quality cannot be assessed prior to germination. Nevertheless, air-dry pea seeds differing in germinability can be divided into three fractions using the method of room temperature phosphorescence (RTP): fraction I, alive high-quality seeds which will produce normal seedlings; fraction II, alive but weak seeds which will produce morphologically abnormal seedlings or will not germinate at all; and fraction III, dead seeds. The imbibition patterns of fraction I and fraction II seeds were compared during germination. The water uptake during imbibition was higher in the fraction II seeds than in those of fraction I. A higher respiration rate and greater limitation of oxygen diffusion by seed coats in fraction II seeds induced oxygen deficiency, which can be considered as hypoxic conditions for embryo. In order to assess the hypoxia level, we developed a non-intrusive luminescent method based on endogenous porphyrin phosphorescence of seeds. Result showed that hypoxia did not appear in fraction I seeds. In fraction II pea seeds, the hypoxia starts to develop after 12-16 h imbibition. When oxygen deficiency enhanced, alive seeds did not germinate as they died due to suffocation during imbibition. If the level of oxygen deficiency was not high (<50 arbitrary units (aU) of porphyrin phosphorescence) and radicles protruded, the emerging seedlings exhibit various morphological defects. Since hypoxia did not impair DNA replication (2C-4C) prior to radicle protrusion, it did not directly contribute to morphological abnormalities in seedlings.

To date, one asparagine synthetase (AS) mRNA has been identified in Nicotiana tabacum. Our results point out that this gene encodes an actual AS protein. This proposal is supported by the following facts. (i) Blast analysis indicates that tobacco AS gene shows increased percent identity with several AS genes of different plant species. (ii) Leaf and root AS mRNA levels were inversely correlated with soluble carbohydrate concentrations, both in the presence of light and in darkness, and the addition of exogenous sucrose repressed the root AS gene expression. (iii) In light, a positive correlation between AS transcript levels and ammonium content was observed in both leaves and roots. (iv) A direct correlation between leaf AS gene expression and asparagine (Asn) concentration has been found. Furthermore, quantitative real-time PCR analyses showed that this gene expression was increased under boron (B) deficiency in roots, but not in leaves. Interestingly, soluble B concentration in roots of plants subjected to B deficiency was significantly lower than that of control plants. However, in leaves, there was no significant difference in soluble B content between both B treatments. We discuss the possible role of soluble B deficiency in the regulation of AS gene expression in tobacco roots. ß addresses: vmbeagal@upo.es (V.M. Beato), jrexben@upo.es (J. Rexach), mtnavgoc@upo.es (M.T. Navarro-Gochicoa), jjcamcri@upo.es (J.J. Camacho-Cristó bal), mbherrod@upo.es (M.B. Herrera-Rodríguez), maldonado@us.es (J.M. Maldonado), agonfon@upo.es (A. Gonzá lez-Fontes

Respective roles of the glutamine synthetase/glutamate synthase cycle and glutamate dehydrogenase in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula Abstract Our objective was to determine the respective roles of the couple glutamine synthetase/glutamate synthase (GS/GOGAT) and glutamate dehydrogenase (GDH) in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula Gaertn. For this aim, amino acids were analyzed by HPLC and changes in gene expression of several enzymes involved in N and C metabolism were studied by real-time quantitative reverse transcription-polymerase chain reaction. Among the enzymes studied, GDH showed the highest increase in gene expression (80-fold), specifically in the embryo axis and concomitant with the increase in ammonium content during post-germinative growth. In cotyledons, GDH gene expression was very low. Although in vitro GDH aminating activity was several times higher than its deaminating activity, in vivo 15 NH 4 incorporation into amino acids was completely inhibited by methionine sulfoximine, a GS inhibitor, indicating that GDH is not involved in ammonium assimilation/detoxification. Changes in the expressions of GS and GOGAT isoforms revealed that GS1b (EC 6.3.1.2) in concert with NADH-dependent GOGAT (EC 1.4.1.14) constitute the major route of assimilation of ammonium derived from reserve mobilization and glutamic acid/glutamine syn-thesis in germinating M. truncatula seeds. However, during post-germinative growth, although germination was held in darkness, expression of GS2 and Fd-GOGAT (EC 1.4.7.1) increased and expression of GS1b decreased in cotyledons but not in the embryo axis. 2-Oxoglutarate, the substrate of the transamination reaction, was provided by the cytosolic isoform of isocitrate dehydrogenase (EC 1.1.1.42). We suggest that GDH during post-germinative growth, specifically in the developing embryo axis, contributes to ammonium delivery to GS for glutamine synthesis in the absence of primary NO 3 ) assimilation. Interestingly, this reaction also produces reducing power (NADH) in organs deprived of photosynthesis. Keywords Amino acid metabolism AE Germination AE Glutamate dehydrogenase AE Glutamine synthetase/ glutamate synthase AE Medicago AE Post-germinative growth Abbreviations EST: Expressed sequence tag AE Fd-GOGAT: Ferredoxin-dependent glutamate synthase AE GDH: Glutamate dehydrogenase AE GS: Glutamine synthetase AE GS1a, GS1b: Cytosolic GS AE GS2: Chloroplastic GS AE cICDH: Cytosolic isocitrate dehydrogenase AE mIDH: Mitochondrial isocitrate dehydrogenase AE MSC27: Medicago sativa cDNA 27 AE MSX: Methionine sulfoximine AE NADH-GOGAT: NADH-dependent glutamate synthase AE PEPc: Phosphoenolpyruvate carboxylase