Enzymes of nitrogen assimilation in maize roots

The intracellular distribution of enzymes involved in the Crassulacean acid metabolism (CAM) has been studied in Bryophyllum calycinum Salisb. and Crassula lycopodioides Lam. After separation of cell organelles by isopycnic centrifugation, enzymes of the Crassulacean acid metabolism were found in the following cell fractions: Phosphoenolpyruvate carboxylase in the chloroplasts; NAD-dependent malate dehydrogenase in the mitochondria and in the supernatant; NADP-dependent malate dehydrogenase and phosphoenolpyruvate carboxykinase in the chloroplasts; NADP-dependent malic enzyme in the supernatant and to a minor extent in the chloroplasts; NAD-dependent malic enzyme in the supernatant and to some degree in the mitochondria; and pyruvate; orthophosphate dikinase in the chloroplasts. The activity of the NAD-dependent malate dehydrogenase was due to three isoenzymes separated by (NH₄)₂SO₄ gradient solubilization. These isoenzymes represented 17, 78, and 5% of the activity recovered, respectively, in the order of elution. The isoenzyme eluting first was associated with the mitochondria and the second isoenzyme was of cytosolic origin, while the intracellular location of the third isoenzyme was probably the peroxisome. Based on these findings, the metabolic path of Crassulacean acid metabolism within cells of CAM plants is discussed. New address: Institut für Pflanzenphysiologie und Zellbiologie, Freie Universität Berlin, Königin-Luise-Straße 12-16a. D-1000 Berlin 33     

Rapid synthesis of glutamine during the initial period of ammonia assimilation in roots of barley plants

After 1 hr of ammonia assimilation in barley plants, glutaminecontent in the roots increased 20 fold its original content,whereas contents of glutamic and aspartic acid decreased toa remarkable extent. These findings indicate a rapid synthesisof glutamine during the initial period of ammonia assimilationin roots. Results also suggest that glutamine synthesis fromglutamic acid initially occurring in the roots, is a primaryreaction of ammonia assimilation (Received May 11, 1971; )     

Synthesis of glutamine and glutamate by intact bundle-sheath cells of maize (Zea mays L.)

Intact bundle-sheath cells with functional plasmodesmata were isolated from leaves of Zea mays L. cv. Mutin, and the capacity of these cells to synthesize glutamine and glutamate was determined by simulating physiological substrate concentrations in the bathing medium. The results show that glutamine synthetase can operate at full rate in the presence of added 8 mM ATP. At lower concentrations of ATP a higher rate of glutamine synthesis was found in the light than in darkness. Glutamate-synthase activity, on the other hand, was strictly light dependent. It appears that in bundle-sheath cells of maize the nitrate-assimilatory capacities of glutamine synthetase (located mainly in the cytosol) and of glutamate synthase (located in the stroma) are high enough to meet the demands of whole maize leaves.Abbreviations Gln glutamine - Glu glutamate - GOGAT glutamate synthase - GS glutamine synthetase - 2-OG 2-oxoglutarate This work was supported by the Bundesminister für Forschung und Technologie (0319296A). We thank Mr. Bernd Raufeisen for the art work of Fig. 1.     

Conversion of 15N-ammonium in forest soils

To determine the fate of atmospheric ammonium in forest soils, one calcareous and two acid forest soils were incubated with ¹⁵N ammonium. In the calcareous soil about 65% of the applied ¹⁵N-ammonium was recovered as nitrate after 98 days of incubation, whereas in the acid soils less than 10% of the ¹⁵N-ammonium was converted to nitrate. In all soils a large proportion of the ¹⁵N was incorporated in organic nitrogen compounds. This incorporation limits the use of ¹⁵N tracers for the elucidation of the fate of atmospheric ammonium in soils.     

Silicon improves salinity tolerance and affects ammonia assimilation in maize roots

The present study was conducted to evaluate the effect of different salt concentrations (50 and 200 mM NaCl) on growth, permeability properties (electrolyte leakage, cell viability) and activity of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in roots of maize seedlings. Both salt concentrations significantly affected growth and permeability properties of maize seedling roots and this negative effect increased with concentration of salt and duration of experiments. On the other hand salinity induced only small changes in the activities of GS and GDH, usually small increase in the activity was observed. To characterise the possible protective effect of silicon (Si) on maize roots exposed to saline stress, different concentrations of Si were simultaneously applied to both, low (50 mM) and high (200 mM) salt concentrations. Possible protective effects of Si on studied parameters were analysed in time range of 3 days treatment with the most positive effect on salt-induced root growth inhibition at high salt concentration and electrolyte leakage. The results show significant increase in GDH activity under all the tested conditions, although the mechanisms underlying this increase have not been elucidated. The results indicate that silicon may ameliorate the salt-induced root growth inhibition and increase the plant vigour at stressful conditions.     

Modelling postsilking nitrogen fluxes in maize (Zea mays) using 15N-labelling field experiments

In maize (Zea mays), nitrogen (N) remobilization and postflowering N uptake are two processes that provide amino acids for grain protein synthesis. To study the way in which N is allocated to the grain and to the stover, two different 15N-labelling techniques were developed. 15NO(3-) was provided to the soil either at the beginning of stem elongation or after silking. The distribution of 15N in the stover and in the grain was monitored by calculating relative 15N-specific allocation (RSA). A nearly linear relationship between the RSA of the kernels and the RSA of the stover was found as a result of two simultaneous N fluxes: N remobilization from the stover to the grain, and N allocation to the stover and to the grain originating from N uptake. By modelling the 15N fluxes, it was possible to demonstrate that, as a consequence of protein turnover, a large proportion of the amino acids synthesized from the N taken up after silking were integrated into the proteins of the stover, and these proteins were further hydrolysed to provide N to the grain.     

Carotenoids in roots indicated the level of stress induced by mannitol and sodium azide treatment during the early stages of maize germination

Chemical mutagens, such as sodium azide, have attracted the interest of plant breeders. Azide creates DNA point mutations and affects plant growth and development, disturbs metabolic activity and inhibits protein and DNA replication, whereas mannitol is used to simulate drought stresses in tissue culture. To identify biochemical markers for stress tolerance, maize seeds were germinated under mannitol and sodium azide induced stress in controlled conditions for 7 days. Then levels of chlorophyll, carotenoids, phenolics and aldehydes produced were subsequently determined. Germination percentage was not affected by either mannitol or sodium azide and was always above 85%. However, total fresh weight decreased by 50% with the application of 153.4 mM mannitol and 0.26 mM azide in combination. This treatment significantly reduced plantlet growth from 0.94 g in the control to 0.53 g in the treated materials. Root weight reduced by 68.1%, cotyledons by 14.3%, stems by 65.0% and leaves by 70.0% in treated samples. The level of carotenoids in roots was the clearest biochemical indicator of stress produced by the mannitol and sodium azide treatment. Carotenoids increased from 0.01 µg g^??1 fresh weight in the control to 9.03 µg g^??1 fresh weight in the treated materials. A large-scale seed treatment with mannitol and sodium azide was carried out. 2296 seeds were placed in magenta containers with 153.4 mM mannitol and 0.26 mM NaN₃. At 7 days of germination, the heaviest seedlings (450) (450/2296?=?20%) were transferred to soil environment. Forty-two plants (42/450?=?9.3%) were off-type phenotypes at 45 days. Genetic variants may have been obtained following the novel procedure described here which combines chronic treatment with sodium azide and selection pressure with mannitol to simulate drought conditions.     

The involvement of glutamine synthetase/glutamate synthase in ammonia assimilation by Aspergillus nidulans

Wild-type Aspergillus nidulans grew equally well on NH4Cl, KNO3 or glutamine as the only nitrogen source. NADP+-dependent glutamate dehydrogenase (EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) activities varied with the type and concentration of nitrogen source supplied. Glutamate synthase (GOGAT) activity (EC 1.4.7.1) was detected but it was almost unaffected by the type and concentration of nitrogen source supplied. Ion exchange chromatography showed that the GOGAT activity was due to a distinct enzyme. Azaserine, an inhibitor of the GOGAT reaction, reduced the glutamate pool by 60%, indicating that GOGAT is involved in ammonia assimilation by metabolizing the glutamine formed by GS.     

Evidence of ammonium assimilation via the glutamine synthetase-glutamate synthase system in rice seedling roots

When rice seedling roots were fed ¹⁵N-ammonium for 1 hr, theamide nitrogen of glutamine showed the highest ¹⁵N abundance.Moreover, glutamine amino, glutamic acid, aspartic acid andalanine showed higher ¹⁵N abundance than ammonium did. In roots whose GS activity was inhibited with MS, both the amountof ammonium and its ¹⁵N abundance were increased. In contrast,both the amount of all examined amino acids containing glutamicacid and their ¹⁵N abundance decreased in roots whose GS activitywas inhibited. From these results, it could be concluded thatthe first step of ammonium assimilation in rice seedling rootswas mainly glutamine synthesis by GS and the second was glutamicacid formation by the GOGAT system. The results of an experiment using ¹⁵N glutamine also supportedthis conclusion. (Received February 23, 1977; )     

15N-ammonium and 15N-nitrate uptake of a 15-year-old Picea abies plantation

Throughfall nitrogen of a 15-year-old Picea abies (L.) Karst. (Norway spruce) stand in the Fichtelgebirge, Germany, was labeled with either ¹⁵N-ammonium or ¹⁵N-nitrate and uptake of these two tracers was followed during two successive growing seasons (1991 and 1992). ¹⁵N-labeling (62 mg ¹⁵N m^-2 under conditions of 1.5 g N m^-2 atmospheric nitrogen deposition) did not increase N concentrations in plant tissues. The ¹⁵N recovery within the entire stand (including soils) was 94%±6% of the applied ¹⁵N-ammonium tracer and 100%±6% of the applied ¹⁵N-nitrate tracer during the 1st year of investigation. This decreased to 80%±24% and 83%±20%, respectively, during the 2nd year. After 11 days, the ¹⁵N tracer was detectable in 1-year-old spruce needles and leaves of understory species. After 1 month, tracer was detectable in needle litter fall. At the end of the first growing season, more than 50% of the ¹⁵N taken up by spruce was assimilated in needles, and more than 20% in twigs. The relative distribution of recovered tracer of both ¹⁵N-ammonium and ¹⁵N-nitrate was similar within the different foliage age classes (recent to 11-year-old) and other compartments of the trees. ¹⁵N enrichment generally decreased with increasing tissue age. Roots accounted for up to 20% of the recovered ¹⁵N in spruce; no enrichment could be detected in stem wood. Although ¹⁵N-ammonium and ¹⁵N-nitrate were applied in the same molar quantities (¹⁵NH ₄ ⁺ : ¹⁵NO ₃ ^- =1:1), the tracers were diluted differently in the inorganic soil N pools (¹⁵NH ₄ ⁺ /NH ₄ ⁺ : ¹⁵NO ₃ ^- /NO ₃ ^- =1:9). Therefore the measured ¹⁵N amounts retained by the vegetation do not represent the actual fluxes of ammonium and nitrate in the soil solution. Use of the molar ammonium-to-nitrate ratio of 9:1 in the soil water extract to estimate ¹⁵N uptake from inorganic N pools resulted in a 2–4 times higher ammonium than nitrate uptake by P. abies.     

A comparison of glutamate synthase obtained from maize endosperms and roots

Glutamate synthase (EC 2.6.1.53) has been examined in developing endosperms and roots of maize. KCl is required for maximum activity in each tissue. The effect with KCl is seen with buffer strength of 25 to 100 millimolar in the assay. The optimum concentration for the enzyme from endosperm is 20 millimolar and for the enzyme from root tissue the saturating concentration is about 20 millimolar. In root material the enzyme is labile but activity can be restored if KCl is added to the assay. Divalent cations such as Mg²⁺ or Mn²⁺ also activate the enzyme to some extent.     

Distinctive characteristics of protein synthesis in maize embryos during the early stages of germination

Quiescent maize embryos were found to contain significant amounts of poly-A-rich pre-formed RNA. ¹⁴C-amino acid incorporation into trichloroacetic acid precipitable material was detected at slow rate at the begining of imbibition and fastly increased near 18 to 24 h. Polysomal formation was measured during this period. Addition of - amanitin to the incubation system at two 6h-pulse periods showed significant inhibition of the ¹⁴C-amino acid incorporation for the 18–24 h-period, but not for the 0–6 h-period.     

Seedling age and cytokinin effects on glutamate dehydrogenase activity and nitrogen assimilation in maize leaves

Glutamate dehydrogenase (GDH) activity, protein and total nitrogen contents in the secondary leaves of maize(Zea mays L. cv. Ganga Safed-2) seedlings increased during early seedling growth and then declined after reaching a peak level at either 10 d (GDH) or 12 d (metabolites). While the effect of kinetin on enzyme activity was statistically insignificant, benzyladenine supplied with nutrient solution increased GDH activity in secondary leaves of both 10-d as well as 14-d seedlings. However, both growth regulators increased the contents of total soluble proteins, total nitrogen, chlorophyll(a+b) and carotenoids in both 10 and 14-d old leaves.     

Effect of salicylic acid on nitrite reductase and glutamate dehydrogenase activities in maize roots

The effects of 0.01 to 5 m M salicyclic acid on the increase in nitrite reductase or glutamate dehydrogenase activities in maize roots by nitrate or ammonium respectively, were examined. Nitrite reductase activity was inhibited by the highest concentration of the acid. The activity of NADH-glutamate dehydrogenase was stimulated slightly (but consistently) by the lowest concentration and was inhibited by higher concentrations. Total protein content was also inhibited at high concentrations. When the crude enzyme extract was stored at 25°C in light, the glutamate dehydrogenase activity in the control decreased after 4 h of incubation. Low concentrations of the acid had no effect on this decrease but higher concentration accelerated the process. The divalent cations Caz²⁺, Mn²⁺, Mg²⁺ and Zn²⁺ protected against loss of enzyme activity during storage, both in the absence and presence of the acid. The inhibitory effect of 5 m M salicylic acid on glutamate dehydrogenase activity is apparent due to interference with the activity of the enzyme rather than with its synthesis.     

Involvement of the glutamine synthetase/glutamate synthase pathway in ammonia assimilation by the wood-rotting fungusPleurotus ostreatus

The mycelium of the wood-rotting fungus,P. ostreatus, contains NAD-dependent glutamate synthase inhibited by azaserine.l-Glutamine andl-glutamate are the most important free amino acids in the mycelium. Feeding of the mycelium with nitrogenous substrates showed thatl-glutamate,l-aspartate andl-alanine are interconnected by way of transaminases. After the inhibition of glutamine synthetase by methionine-S-sulfoximine the synthesis ofl-glutamate was inhibited and the level of all free amino acids decreased. The¹⁵N-NMR spectra of mycelia after the addition of¹⁵NH₄Cl confirmed that the GS/GOGAT is the only pathway of ammonia assimilation inP. ostreatus and NAD-glutamate dehydrogenase should be the deaminating enzyme.     

A kinetic study of the assimilation of 15N-labelled ammonium in rice seedling roots

The pathway of ammonium nitrogen assimilation, its incorporationinto amino acids and synthesis of protein was studied with theaid of nitrogen-15. The analysis of ¹⁵N involves the use ofoptical emission spectrometry. Kinetic analysis of nitrogen assimilation by the roots indicatesthat glutamine and glutamic acid were the primary products ofammonium assimilation. Possibly some of the amino acids, suchas aspartic acid and alanine received their amino nitrogen directlyfrom free ammonia in the roots. Amino groups were transformedinto other amino acids from these primary products, especiallyfrom glutamic acid through transamination. (Received April 1, 1974; )     

Regulation of mitochondrial glutamine/glutamate metabolism by glutamate transport: studies with (15)N

We focused on the role of plasma membrane glutamate uptake in modulating the intracellular glutaminase (GA) and glutamate dehydrogenase (GDH) flux and in determining the fate of the intracellular glutamate in the proximal tubule-like LLC-PK(1)-F(+) cell line. We used high-affinity glutamate transport inhibitors D-aspartate (D-Asp) and DL-threo-beta-hydroxyaspartate (THA) to block extracellular uptake and then used [(15)N]glutamate or [2-(15)N]glutamine to follow the metabolic fate and distribution of glutamine and glutamate. In monolayers incubated with [2-(15)N]glutamine (99 atom %excess), glutamine and glutamate equilibrated throughout the intra- and extracellular compartments. In the presence of 5 mM D-Asp and 0.5 mM THA, glutamine distribution remained unchanged, but the intracellular glutamate enrichment decreased by 33% (P < 0.05) as the extracellular enrichment increased by 39% (P < 0.005). With glutamate uptake blocked, intracellular glutamate concentration decreased by 37% (P < 0.0001), in contrast to intracellular glutamine concentration, which remained unchanged. Both glutamine disappearance from the media and the estimated intracellular GA flux increased with the fall in the intracellular glutamate concentration. The labeled glutamate and NH formed from [2-(15)N]glutamine and recovered in the media increased 12- and 3-fold, respectively, consistent with accelerated GA and GDH flux. However, labeled alanine formation was reduced by 37%, indicating inhibition of transamination. Although both D-Asp and THA alone accelerated the GA and GDH flux, only THA inhibited transamination. These results are consistent with glutamate transport both regulating and being regulated by glutamine and glutamate metabolism in epithelial cells.