Role of asparagine in the photorespiratory nitrogen metabolism of pea leaves

In pea leaves, much of the metabolism of imported asparagine is by transamination. This activity was previously shown to be localized in the peroxisomes, suggesting a possible connection between asparagine and photorespiratory nitrogen metabolism. This was investigated by examination of the transfer of ¹⁵N from the amino group of asparagine, supplied via the transpiration stream, in fully expanded pea leaves. Label was transferred to aspartate, glutamate, alanine, glycine, serine, ammonia, and glutamine (amide group). Under low oxygen (1.8%), or in the presence of α-hydroxy-2-pyridine methanesulfonic acid (an inhibitor of glycolate oxidase, a step in the photorespiratory formation of glyoxylate), there was a substantial (60-80%) decrease in transfer of label to glycine, serine, ammonia, and glutamine. Addition of isonicotinyl hydrazide (an inhibitor of formation of serine from glycine) caused a 70% decrease in transfer of asparagine amino nitrogen to serine, ammonia, and glutamine, while a 4-fold increase in labeling of glycine was observed. The results demonstrate the involvement of asparagine in photorespiration, and show that photorespiratory nitrogen metabolism is not a closed cyclic process.     

Utilization of the amide groups of asparagine and 2-hydroxysuccinamic Acid by young pea leaves

The fate of nitrogen originating from the amide group of asparagine in young pea leaves (Pisum sativum) has been studied by supplying [¹⁵N-amide]asparagine and its metabolic product, 2-hydroxysuccinamate (HSA) via the transpiration stream. Amide nitrogen from asparagine accumulated predominantly in the amide group of glutamine and HSA, and to a lesser extent in glutamate and a range of other amino acids. Treatment with 5-diazo,4-oxo-L-norvaline (DONV) a deamidase inhibitor, caused a decrease in transfer of label to glutamine-amide. Virtually no ¹⁵N was detected in HSA of leaves supplied with asparagine and the transaminase inhibitor aminooxyacetate. When [¹⁵N]HSA was supplied to pea leaves, most of the label was also found in the amide group of glutamine and this transfer was blocked by the addition of methionine sulfoximine, which caused a large increase in NH₃ accumulation. DONV was not specific for asparaginase, and inhibited the deamidation of HSA, causing a decrease in transfer of ¹⁵N into glutamine-amide, NH₃, and other amino acids. It is concluded from these results that use of the amide group of asparagine as a nitrogen source for young pea leaves involves deamidation of both asparagine and its transamination product HSA (possibly also oxosuccinamate). The amide group, released as ammonia, is then reassimilated via the glutamine synthetase/glutamate synthase system.     

Amino Acid metabolism in pea leaves : utilization of nitrogen from amide and amino groups of [N]asparagine

The flow of nitrogen from the amino and amide groups of asparagine has been followed in young pea (Pisum sativum CV Little Marvel) leaves, supplied through the xylem with ¹⁵N-labeled asparagine. The results confirm that there are two main routes for asparagine metabolism: deamidation and transamination.

Nitrogen from the amide group is found predominantly in 2-hydroxy-succinamic acid (derived from transamination of asparagine) and in the amide group of glutamine. The amide nitrogen is also found in glutamate and dispersed through a range of amino acids. Transfer to glutamineamide results from assimilation of ammonia produced by deamidation of both asparagine and its transamination products: this assimilation is blocked by methionine sulfoximine. The release of amide nitrogen as ammonia is greatly reduced by aminooxyacetate, suggesting that, for much of the metabolized asparagine, transamination precedes deamidation.

The amino group of asparagine is widely distributed in amino acids, especially aspartate, glutamate, alanine, and homoserine. For homoserine, a comparison of N and C labeling, and use of a transaminase inhibitor, suggests that it is not produced from the main pool of aspartate, and transamination may play a role in the accumulation of homoserine in peas.

     

Nitrogen assimilation in citrus trees

Assimilation of ¹⁵N-ammonium and ¹⁵N-nitrate was examined in 3-year-old satsuma mandarin (Citrus unshiu Marcovitch) trees. Experiments were designed to establish the time course of incorporation of nitrogen just taken up into amino compounds. In fine roots, absorbed ¹⁵N-ammonium was actively incorporated into glutamine and then into glutamic acid and asparagine. When feeding ¹⁵N-nitrate, glutamic acid and asparagine were actively synthesized, but glutamine synthesis was comparatively low as compared with that in ammonium feeding. In current leaves and fruits, a clear difference in the labelling patterns of amino acids was found between the ammonium and nitrate feedings. The amino acid most markedly labelled was asparagine in the ammonium feeding and glutamine in the nitrate feeding. Considering the most heavily labelled component in leaves and fruits, the main form of the nitrogen components transported upward in the xylem was discussed.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch)

The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4-methyleneglutamine. Pulse-feeding nodulated roots with ¹⁵N₂ gas showed asparagine to be the principal nitrogen product exported from N₂-fixing nodules. Maintaining root systems in an N₂-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N₂ fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of ¹⁵N-nitrate, and a ¹⁵N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N₂ or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N₂. Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N₂ and nitrate are being used in peanuts.     

Nitrogen Metabolism of Vicia faba

Vicia faba plants were grown for four and six weeks without externally supplied nitrogen. Some nitrogen was transported to the plant axis from the cotyledons throughout this period, but the amount available was insufficient to support maximum shoot growth. During this period the protein content of the shoot declined whilst the free amino acids, especially aspartic acid, glutamic acid, histamine and the combined pool for threonine, serine, asparagine and glutamine and ammonia, increased in amount. In contrast to the shoot the protein content of the root increased as did their free amino acid content, but the increase in the latter was less than in the shoot and only the combined value for threonine, serine, asparagines and glutamine increased significantly. During tbe last two weeks growth, some soluble non-amino acid compound appeared to donate nitrogen to the pool of free amino acids in the root and shoot.     

Nitrogen nutrition in the cyanobacterium Nostoc ANTH, a symbiotic isolate from Anthoceros: uptake and assimilation of inorganic-n and amino acids

Amino acid uptake and utilization of various nitrogen sources (amino acids, nitrite, nitrate and ammonia) were studied in Nostoc ANTH and i ts mu tant (Het(-)Nif(-)) isolate defective in heterocyst formation and N2-fixation. Both parent and its mutant grew at the expense of glutamine, asparagine and arginine as a source of fixed-nitrogen. Growth was better in glutamine-and asparagine-media as compared to that in arginine media. Glutamine and asparagine repressed heterocyst formation, N2-fixation and nitrate reduction in Nostoc ANTH, but arginine did so only partially. The poor growth in arginine-medium was not due to poor uptake rates, since the uptake rates were not significantly different from those for glutamine or asparagine. The glutamine synthetase activity remained unaffected during cultivation in media containing any one of the three amino acids tested. The uptake of amino acids was substrate-inducible, energy-dependent and required de novo protein synthesis. Nitrate and ammonium repressed ammonium uptake, but did not repress uptake of amino acids. In N2-medium (BG-11(0)), the uptake of ammonium and amino acids in the mutant was significantly higher than its parent strain. This was apparently due to nitrogen limitation since the mutant was unable to fix N2 and the growth medium lacked combined-N.     

Effects of applied nitrogen upon aspects of nitrogen metabolism and transport in developing nodulated winged bean plants

Nodulated winged bean [Psophocarpus tetragonolobus (L.) DC., cv. UPS 122] were grown under constant environmental conditions and supplied with mineral nutrient solution in which nitrogen was absent or was present as nitrate (12 mg N week^-1 plant^-1). Nitrate treatment dramatically promoted plant growth, increased fruit weight 1.6 fold, was necessary for tuberisation and enhanced nodulation. The in vitro accumulation of ¹⁴C into asparagine and aspartate components of excised nodules supplied with exogenous ¹⁴CO₂ and [¹⁴C]-D-glucose was greater for nitrate-treated plants, whilst accumulation into ureides was reduced by nitrate treatment. Levels of amino acids in xylem sap were greater for plants supplied with a complete nutrient solution, than those grown without applied nitrate, particularly for asparagine, glutamine and proline. Xylem ureide levels were greater for plants grown in the absence of supplementary nitrate. Nitrogen accumulated in leaf, stem and petiole, and root nodule tissues for utilisation during fruit development; peak nitrogen levels and time of anthesis were retarded for plants grown without applied nitrate. The shoot ureide content increased during fruiting, coincident with decreases in the total nitrogen content, indicating that ureide pools are not utilised during the early reproductive phase. However ureide reserves, particularly allantoin, were utilised during the later stages of pod fill. Enzyme activity which metabolised asparagine was found throughout the plant and was identified as K⁺-dependent asparaginase (EC 3.5.1.1) and an aminotransferase. Apart from temporal differences in developmental profiles of enzyme activity, the activity of these enzymes and of allantoinase (EC 3.5.2.5) in developing tissues were similar for both treatments. The main differences were greater asparaginase and asparagine:pyruvate aminotransferase activities in root tissues and fruit of nitrate-supplied plants; allantoinase activity in the primary roots of plants grown without nitrate decreased during development, whilst activity in developing tubers (nitrate-supplied plants) increased.     

Characterization of nitrogen relationships between Sorghum bicolor and the root-hemiparasitic angiosperm Striga hermonthica (Del.) Benth. using K15 NO3 as isotopic tracer

The role of the host in the nitrogen nutrition of Striga hermonthica (Del.) Benth. (Scrophulariaceae) parasitic on Sorghum bicolor cv. SH4 Arval has been investigated using (15)N-nitrate as the tracer. It is shown that, when nitrate is absorbed only by the roots of the host plant, a rapid transfer of nitrogen to the parasite can be detected. The xylem sap of S. hermonthica contained approximately equal amounts of nitrate and amino acids, mostly glutamine and asparagine. Infection altered the free amino acid profile of the host tissues, leading notably to a large increase in asparagine and a decrease in glutamine. The haustoria of S. hermonthica, although rich in nitrate, showed a low concentration of free amino acids, particularly lacking in asparagine and glutamine. The roots of S. hermonthica, in contrast, were rich in both asparagine and glutamine while, in the shoots, asparagine constituted 80% of the total FAA pool. Asparagine was also found to be the primary (15)N-enriched amino acid in the shoots of S. hermonthica while, interestingly, it was glutamate that was most strongly enriched in the roots. It is concluded that nitrogen nutrition in S. hermonthica is based on a supply of both nitrate and amino acids from the host. This implies a non-specific transfer in the transpiration stream. Nitrate reduction probably occurs mainly in the leaves of the parasite. Assimilation also occurs in S. hermonthica and excess nitrogen is stored as the non-toxic nitrogen-rich compound, asparagine. This specific trait of nitrogen metabolism of the parasite is discussed in relation to the effect of nitrogen fertilization on reducing infestation.     

The impact of mycorrhizal colonization upon nitrogen source utilization and metabolism in seedlings of Eucalyptus grandis Hill ex Maiden and Eucalyptus maculata Hook

Analysis of soil solution from forest sites dominated by Eucalyptus grandis and Eucalyptus maculata indicates that soluble forms of organic nitrogen (amino acids and protein) are present in concentrations similar to those of mineral nitrogen (nitrate and ammonium). Experiments were conducted to determine the extent to which mycorrhizal associations might broaden nitrogen source utilization in Eucalyptus seedlings to include organic nitrogen. In isolation, species of ectomycorrhizal fungi from northern Australia show varying abilities to utilize mineral and organic forms of nitrogen as sole sources. Pisolithus sp. displayed strongest growth on NH₄⁺, glutamine and asparagine, but grew poorly on protein, while Amanita sp. grew well both on mineral sources and on a range of organic sources (e.g. arginine, asparagine, glutamine and protein). In sterile culture, non-mycorrhizal seedlings of Eucalyptus grandis and Eucalyptus maculata grew well on mineral sources of nitrogen, but showed no ability to grow on sources of organic nitrogen other than glutamine. In contrast, mycorrhizal seedlings grew well on a range of organic nitrogen sources. These observations indicate that mycorrhizal associations confer on species of Eucalyptus the ability to broaden their resource base substantially with respect to nitrogen. This ability to utilize organic nitrogen was not directly related to that of the fungal symbiont in isolation. Seedlings mycorrhizal with Pisolithus sp. were able to assimilate sources of nitrogen (in particular histidine and protein) on which the fungus in pure culture appeared to grow weakly. Experiments in which plants were fed ¹⁵N-labelled ammonium were undertaken in order to investigate the influence of mycorrhizal colonization on the pathway of nitrogen metabolism. In roots and shoots of all seedlings, ¹⁵N was incorporated into the amide group of glutamine, and label was also found in the amino groups of glutamine, glutamic acid, γ-aminobutyric acid and alanine. Mycorrhizal colonization appeared to have no effect on the assimilation pathway and metabolism of [¹⁵N]H₄⁺; labelling data were consistent with the operation of the glutamate synthase cycle in plants infected with either Pisolithus sp. (which in isolation assimilates via the glutamate synthase cycle) or Elaphomyces sp. (which assimilates via glutamate dehydrogenase). It is likely that the control of carbon supply to the mycorrhizal fungus from the host may have a profound effect on both the assimilatory pathway and the range of nitrogen sources that can be utilized by the association.     

Glutamine- and Asparagine-Dependent Protein Synthesis in Maturing Legume Cotyledons Cultured in vitro

Legume cotyledons have been grown in sterile culture and shownto synthesize up to 16 mg protein in 5 d when supplied witheither asparagine or glutamine as sole nitrogen sources. Glutamate,nitrate, and ureides also served as nitrogen sources for proteinsynthesis but to a lesser extent. Methionine sulphoximine and azaserine inhibited asparagine-dependentprotein synthesis suggesting that ammonia is liberated fromasparagine which is reassimilated via glutamine synthetase andglutamate synthase.     

Ammonia Assimilation in the Roots of Nitrate- and Ammonia-Grown Hordeum Vulgare (cv Golden Promise)

¹⁵N kinetic labeling studies were performed on seedlings of Hordeum vulgare L. var. Golden Promise growing under steady state conditions. Patterns of label incorporation in the pools of nitrogen compounds of roots fed [¹⁵N]ammonium were compared with computer-simulated labeling curves. The data were found to be quantitatively consistent with a three-compartment model in which ammonium is assimilated solely into the amide-N of glutamine. Labeling data from roots fed [¹⁵N]nitrate were also found to be at least qualitatively consistent with the assimilation of ammonia into glutamine. Methionine sulfoximine almost completely blocked the incorporation of ¹⁵N label into the amino acid pools of barley roots fed [¹⁵N]nitrate. These observations suggest that ammonia assimilation occurs solely via the glutamine synthetase/glutamate synthase pathway in both nitrate- and ammonia-grown barley roots.     

INFLUENCE OF AMMONIUM AND NITRATE ON THE PROTEIN- AND FREE AMINO ACIDS IN SHOOTS OF WHEAT SEEDLINGS

Weissman , Gerard S. (Rutgers U., Camden, N. J.) Influence of ammonium and nitrate on the protein- and amino acids in shoots of wheat seedlings. Amer. Jour. Bot. 46(5): 339–346. 1959.—Total and protein nitrogen per shoot of wheat seedlings grown with endosperm attached increased at a steady rate during a 96-hr. growth period, and protein nitrogen, as a percentage of total nitrogen, remained constant at about 53%. Total and protein nitrogen concentration was greatest for 24-hr. shoots and declined as the shoots became older. Total and protein nitrogen were determined in 96-hr. shoots of seedlings grown with endosperm attached but also supplied with ammonium, nitrate, or both in the culture solution. Total nitrogen was greatest in shoots supplied with ammonium, but only 38% was in the form of protein. Maximum protein synthesis occurred in shoots grown in both ammonium and nitrate and protein nitrogen as a percentage of total nitrogen approximated that achieved in shoots lacking nitrogen in the culture solution. The protein amino acid composition of 48-, 72-, and 96-hr. shoots was very similar but differed from 24-hr. shoots which contained higher percentages of arginine and lysine and lower percentages of alanine and threonine. This may be correlated with the higher proportion of meristematic cells in 24-hr. shoots. The protein amino acids in shoots grown with ammonium resembled that of shoots lacking nitrogen in the culture solution, but nitrate shoot protein contained a higher percentage of arginine and a lower percentage of lysine. Nitrate may stimulate the formation of enzymes, possibly of a nitrate-reducing system, with high arginine- low lysine content. Free asparagine and glutamine were both at a maximum in ammonium shoots and at a minimum in nitrate shoots, but asparagine predominated in shoots supplied with ammonium while glutamine was greatest in nitrate shoots. Aspartic acid, asparagine, and glutamine appeared to have ammonia-storage functions, but glutamic acid appeared to be primarily concerned with protein synthesis. Amino acid accumulation was greatest in shoots supplied with both ammonium and nitrate. Protein synthesis in these appeared to be limited by inadequate concentrations of glutamic acid and proline. A hypothesis is proposed in explanation of the high glutamic acid concentration in shoots provided with ammonium and nitrate.     

Xylem to phloem transfer of solutes in fruiting shoots of legumes,studied by a phloem bleeding technique

Summary Comparisons were made of the levels of various solutes in xylem (tracheal) sap and fruit tip phloem sap of Lupinus albus (L.) and Spartium junceum (L.). Sucrose was present at high concentration (up to 220 mg ml^-1) in phloem but was absent from xylem whereas nitrate was detected in xylem (up to 0.14 mg ml^-1) but not in phloem. Total amino acids reached 0.5–2.5 mg ml^-1 (in xylem) versus 16–40 mg ml^-1 in phloem. Phloem: xylem concentration ratios for mineral nutrients (K, Na, Mg, Ca, Fe, Zn, Mn, Cu) spanned the range 0.7 to 20, the ratios generally reflecting an element's phloem mobility and its availability to the xylem from the roots.The accessibility of nitrate to xylem and phloem was studied in Lupinus. Increasing the nitrate supply to roots from 100 to 1000 mg NO₃–Nl^-1 increased nitrate spill over into xylem, but nitrate always failed to appear in phloem. However, phloem loading of small amounts of nitrate was induced by feeding 750 or 1000 mg NO₃–Nl^-1 directly to cut shoots via the transpiration stream. Transfer of reduced nitrogen to phloem was demonstrated by feeding ¹⁵NO₃ to shoots and recovering ¹⁵N-enriched amides and amino acids in phloem sap. Increased nitrate supply to roots led to increased amino acid levels in xylem and phloem but did not alter markedly the balance between individual amino acids.The fate of xylem-fed ¹⁴C-labelled asparagine, glutamine and aspartic acid and of photosynthetically fed ¹⁴CO₂ was studied in Spartium, with reference to phloem transport to seeds. Substantial fractions of the ¹⁴C of all sources appeared in non-amino compounds. [¹⁴C]asparagine passed largely in unchanged form to the phloem whereas the ¹⁴C from aspartic acid or glutamine appeared in phloem attached to other amino acids (e.g. asparagine and glutamic acid). Serine, asparagine and glutamine were the main amino compounds labelled in phloem sap after feeding ¹⁴CO₂. The wide distribution of ¹⁴C amongst free and bound amino acids of seeds suggested that extensive metabolism of phloem-borne solutes occurred in the fruits.     

Quantitative changes of free amino acids and amides in barley plants during ammonia and nitrate assimilation

Quantitative changes of free amino acids and amides were investigatedin barley plants supplied with ammonia or nitrate for 1 hr,followed by transfer to nitrogen-deficient medium for 6 hr.Changes in levels of free ammonia and nitrate were closely correlatedwith the synthesis of amino acids and amides. In ammonia-supplied roots, rapid primary synthesis and turnoverof glutamine and glutamate occurred, with glutamine playinga leading role in the assimilation. Decreases of glutamate andaspartate during the initial period suggested that glutamatewas synthesized more slowly than glutamine, and the aspartate-glutamatecycle was in operation. After transfer to N-deficient medium,glutamine was rapidly converted into glutamate, which in turnseemed to undergo transamination to aspartate and alanine. Therewas also a rapid rise in O₂-uptake of roots during the 1 hrsupply of ammonia. In nitrate-supplied plants, most of the absorbed nitrate wastransported to the shoot where it was assimilated into glutamateand aspartate. The amount of glutamine increased continuously,suggesting that it was a primary product of nitrate assimilationin roots. The crucial difference observed between synthesis rates of aminocompounds during the initial periods of the two treatments wasprobably due to a lag in nitrate reduction. We will discuss metabolic phenomena associated with active assimilationof ammonia. (Received October 6, 1971; )     

Amino Acid Metabolism of Lemna minor L. : IV. N-Labeling Kinetics of the Amide and Amino Groups of Glutamine and Asparagine

A serious limitation to the use of N(O,S)-heptafluorobutyryl isobutyl amino acid derivatives in the analysis of ¹⁵N-labeling kinetics of amino acids in plant tissues, is that the amides glutamine and asparagine undergo acid hydrolysis to glutamate and aspartate, respectively, during derivatization. This led us to consider an alternative procedure (G Fortier et al. [1986] J Chromatogr 361: 253-261) for derivatization of glutamine and asparagine with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in pyridine. Gas chromatography-mass spectrometry (electron ionization) yielded fragment ions (M-57) of mass 417 and 431 for the [¹⁴N]asparagine and [¹⁴N]glutamine derivatives, respectively, suitable for monitoring unlabeled, single-¹⁵N- and double-¹⁵N-labeled amide species from the ion clusters at mass to charge ratio (m/z) 415 to 423 for asparagine, and m/z 429 to 437 for glutamine. From separate analyses of the specific isotope abundance of the amino-N groups of asparagine and glutamine as their N-heptafluorobutyryl isobutyl derivatives, the specific amide-[¹⁵N] abundance of these amino acids was determined. We demonstrate that this approach to ¹⁵N analysis of the amides can yield unique insights as to the compartmentation of asparagine and glutamine in vivo. The ratios of unlabeled:single-¹⁵N:double-¹⁵N-labeled species are highly diagnostic of the relative sizes and turnover of metabolically active and inactive pools of the amides and their precursors. Kinetic evidence is presented to indicate that a significant proportion (approximately 10%) of the free asparagine pool may be metabolically inactive (vacuolar). If the amide group of asparagine is derived exclusively from glutamine-amide, then asparagine must be synthesized in a compartment of the cell in which both glutamine-amide and aspartate are more heavily labeled with ¹⁵N than the bulk pools of these amino acids. This compartment is presumably the chloroplast. The transaminase inhibitor aminooxyacetate is shown to markedly inhibit amino acid synthesis; several amino acid pools accumulated in the presence of aminooxyacetate and [¹⁵N]H₄⁺ are ¹⁴N-enriched and must be derived primarily from protein turnover.     

Nitrogen Assimilation in Mycorrhizas : Ammonium Assimilation in the N-Starved Ectomycorrhizal Fungus Cenococcum graniforme

Ammonium assimilation was followed in N-starved mycelia from the ectomycorrhizal Ascomycete Cenococcum graniforme. The evaluation of free amino acid pool levels after the addition of 5 millimolar NH₄⁺ indicated that the absorbed ammonium was assimilated rapidly. Post-feeding nitrogen content of amino acids was very different from the initial values. After 8 hours of NH₄⁺ feeding, glutamine accounted for the largest percentage of free amino acid nitrogen (43%). The addition of 5 millimolar methionine sulfoximine (MSX) to NH₄⁺-fed mycelia caused an inhibition of glutamine accumulation with a corresponding increase in glutamate and alanine levels.

Using ¹⁵N as a tracer, it was found that the greatest initial labeling was into glutamine and glutamate followed by aspartate, alanine, and ornithine. On inhibiting glutamine synthetase using MSX, ¹⁵N enrichment of glutamate, alanine, aspartate, and ornithine continued although labeling of glutamine was quite low. Moreover, the incorporation of ¹⁵N label in insoluble nitrogenous compounds was lower in the presence of MSX. From the composition of free amino acid pools, the ¹⁵N labeling pattern and effects of MSX, NH₄⁺ assimilation in C. graniforme mycelia appears to proceed via glutamate dehydrogenase pathway. This study also demonstrates that glutamine synthesis is an important reaction of ammonia utilization.

     

Concentrations of sucrose and nitrogenous compounds in the apoplast of developing soybean seed coats and embryos

The apoplast of developing soybean (Glycine max cv Hodgson) embryos and seed coats was analyzed for sucrose, amino acids, ureides, nitrate, and ammonia. The apoplast concentration of amino acids and nitrate peaked during the most rapid stage of seed filling and declined sharply as the seed attained its maximum dry weight. Amino acids and nitrate accounted for 80 to 95% of the total nitrogen, with allantoin and allantoic acid either absent or present in only very small amounts. Aspartate, asparagine, glutamate, glutamine, serine, alanine, and γ-aminobutyric acid were the major amino acids, accounting for over 70% of the total amino acids present. There was a nearly quantitative conversion of glutamine to glutamate between the seed coat and embryo, most likely resulting from the activity of glutamate synthase found to be present in the seed coat tissue. This processing of glutamine suggests a partly symplastic route for solutes moving from the site of phloem unloading in the seed coat to the embryo.     

Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.Abbreviations SB-P photoautotrophic soybean cells (no sucrose, high CO₂, high light) - SB-M photomixotrophic soybean cells (1% w/v sucrose, high light) - SB-H heterotrophic soybean cells (3% sucrose, dark)