Inhibition of nitrate uptake by aluminium in maize

Experiments with two maize (Zea mays L.) hybrids were conducted to determine (a) if the inhibition of nitrate uptake by aluminium involved a restriction in the induction (synthesis/assemblage) of nitrate transporters, and (b) if the magnitude of the inhibition was affected by the concurrent presence of ambient ammonium. At pH 4.5, the rate of nitrate uptake from 240 μM NH₄NO₃ was maximally inhibited by 100 μM aluminium, but there was little measurable effect on the rate of ammonium uptake. Presence of ambient aluminium did not eliminate the characteristic induction pattern of nitrate uptake upon first exposure of nitrogen-depleted seedlings to that ion. Removal of ambient aluminium after six hours of induction resulted in recovery within 30 minutes to rates of nitrate uptake that were similar to those of plants induced in absence of aluminium. Addition of aluminium to plants that had been induced in absence of aluminium rapidly restricted the rate of nitrate uptake to the level of plants that had been induced in the presence of aluminium. The data are interpreted as indicating that aluminium inhibited the activity of nitrate transporters to a greater extent than the induction of those transporters. When aluminium was added at initiation of induction, the effect of ambient ammonium on development of the inhibition by aluminium differed between the two hybrids. The responses indicate a complex interaction between the aluminium and ammonium components of high acidity soils in their influence on nitrate uptake. ei]{gnA C}{fnBorstlap}     

The effects of nitrate:ammonium ratios and nitrapyrin on the nitrogen response ofZea mays L. under field conditions

Five nitrate:ammonium ratios at two N-levels were tested with and without nitrapyrin [2 chloro-6-(trichloromethyl) pyridine] for grain production on a sandy soil. Treatments were applied to field maize as nutrient solutions, in one application, six weeks after planting. Nytrapyrin resulted in an increase in grain yield at a nitrate:ammonium ratio of 1:3 but in a decrease at a 0:1 ratio. The optimum nitrate:ammonium ratio was close to 1:3 with nitrapyrin and close to 3:1 without nitrapyrin. Nitrapyrin had an effect on NH₄ ⁺-N in the topsoil and NO₃ ⁻-N in the subsoil at 70 days after application. Interactions of nitrate:ammonium ratios and N-levels were shown for leaf N concentration, soil mineral N and soil pH.     

Purification and characterisation of glutamine synthetase fromNocardia corallina

Glutamine synthetase (GS) (EC 6.3.1.2) has been purified 67-fold fromNocardia corallina. The apparentM _r of the GS subunit was approximately 56,000. Assuming the enzyme is a typical dodecamer this indicates a particle mass for the undissociated enzyme of 672,000. The GS is regulated by adenylylation and deadenylylation, and subject to feedback inhibition by alanine and glycine. The pH profiles assayed by the -glutamyl transferase method were similar for NH₄ ⁺-treated and untreated cell extracts and an isoactivity point was not obtained from these curves. GS activity was repressed by (NH₄)₂SO₄ and glutamate. Cells grown in the presence of glutamine, alanine, proline and histidine had enhanced levels of GS activity. The GS ofN. corallina cross-reacted with antisera prepared against GS from a Gram-negativeThiobacillus ferrooxidans strain but not with antisera raised against GS from a Gram-positiveClostridium acetobutylicum strain.     

Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.Abbreviation GS glutamine synthetase     

Differences among maize cultivars in the utilization of soil nitrate and the related losses of nitrate through leaching

In a 2-year field experiment conducted on a Gleyic Luvisol in Stuttgart-Hohenheim one experimental and nine commercial maize cultivars were compared for their ability to utilize soil nitrate and to reduce related losses of nitrate through leaching. Soil nitrate was monitored periodically in CaCl₂ extracts and in suction cup water. Nitrate concentrations in suction water were generally higher than in CaCl₂ extracts. Both methods revealed that all cultivars examined were able to extract nitrate down to a soil depth of at least 120 cm (1988 season) or 150 cm (1987 season). Significant differences among the cultivars existed in nitrate depletion particularly in the subsoil. At harvest, residual nitrate in the upper 150 cm of the profile ranged from 73–110 kg N ha^–1 in 1987 and from 59–119 kg N ha^–1 in 1988. Residual nitrate was closely correlated with nitrate losses by leaching because water infiltration at 120 cm soil depth started 4 weeks after harvest (1987) or immediately after harvest (1988) and continued until early summer of the following year. The calculated amount of nitrate lost by leaching was strongly influenced by the method of calculation. During the winter of 1987/88 nitrate leaching ranged from 57–84 kg N ha^–1 (suction cups) and 40–55 kg N ha^–1 (CaCl₂ extracts), respectively. The corresponding values for the winter of 1988/89 were 47–79 and 20–39 kg N ha^–1, respectively. ei]Section editor: B E Clothier     

Appearance of nitrate reductase, nitrite reductase and glutamine synthetase in different organs of the Scots pine (Pinus sylvestris) seedling as affected by light, nitrate and ammonium

Appearance of nitrate reductase (NR, EC 1.6.6.1–3), nitrite reductase (NiR, EC 1.7.7.1) and glutamine synthetase (GS, EC 6.3.1.2) under the control of nitrate, ammonium and light was studied in roots, hypocotyls and needles (cotyledonary whorl) of the Scots pine ( Pinus sylvestris L.) seedling. It was found that appearance of NiR was mainly controlled by nitrate whereas appearance of GS was strongly controlled by light. In principle, the NR activity level showed the same dependency on nitrate and light as that of NiR. In the root, both nitrate and ammonium had a stimulatory effect on GS activity whereas in the whorl the induction was minor. The level of NiR (NR) activity is high in the root and hypocotyl and low in the cotyledonary whorl, whereas the GS activity level per organ increases strongly from the root to the whorl. Thus, in any particular organ the operation of the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle is not closely connected to the operation of the nitrate reduction pathway. The strong control of GS/GOGAT by light and the minor sensitivity to induction by nitrate or ammonium indicate a major role of the GS/GOGAT cycle in reassimilation of endogeniously generated ammonium.     

Glutamine synthetase and arginine inhibition of nitrate reductase activity in Anabaena cycadeae

Wild-type Anabaena cycadeae with normal glutamine synthetase (GS) activity utilized arginine as sole N source whereas a mutant strain lacking GS activity did not. Nitrate reductase (NR) activity, higher in the mutant strain than the wild-type strain, was inhibited by arginine though arginine-dependent NH ₄ ⁺ generation was higher in the mutant strain than in the wild-type. This suggests that (1) NR activity is NO _inf3 ^sup- -inducible and arginine-repressible; and (2) while GS activity is required for the assimilation of arginine as sole N-source, it is not required for arginine inhibition of NR activity.S. Singh was with the Department of Biochemistry, North-Eastern Hill University, Shillong-793014, India, and is now with P.S. Bisen at the Department of Microbiology, Barkatullah University, Bhopal-462026, India     

Cloning of the glutamine synthetase gene fromspirulina platensis

An 8 Kilobase-pair (Kbp) HindIII fragment containing the coding sequence forSpirulina platensis glutamine synthetase [EC 6.3.1.1.] has been identified utilizing a probe derived fromAnabaena 7120 and cloned in the vector pAT153.     

Intracellular localization of glutamine synthetase in Chlorogloeopsis fritschii

After differential centrifugation of cell-free extracts of Chlorogloeopsis fritschii, 71% of the original glutamine synthetase (GS) activity was associated with the thylakoids, while little activity was detected in the cytoplasmic membranes. Monospecific antiserum to a purified GS inhibited 88% of the enzyme activity in solubilized thylakoid membranes. An antiserum raised against thylakoids gave 81% inhibition. However, using intact thylakoid membranes, only 7% inhibition was obtained with the GS antiserum, indicating that GS is located inside the thylakoid membranes.The author is with the Department of Biological Sciences, University of Science and Technology, Irbid, Jordan     

Expression of glutamine synthetase genes in roots and nodules of Phaseolus vulgaris following changes in the ammonium supply and infection with various Rhizobium mutants

In this paper we have examined whether the four glutamine synthetase (gln) genes, expressed in roots and nodules of Phaseolus vulgaris are substrate-inducible by ammonium. Manipulation of the ammonium pool in roots, through addition and removal of exogenous ammonium, did not elicit any changes in the abundances of the four mRNAs thus suggesting that the gln genes in roots of this legume are neither substrate-inducible by ammonium nor derepressed during nitrogen starvation. In nodules the effect of the ammonium supply on expression of the gln genes has been examined by growing nodules under argon/oxygen atmospheres, or with a number of Fix^- Rhizobium mutants, and following addition of exogenous ammonium. The results of these experiments suggest that the expression of the gln- gene, which is strongly induced during nodule development, is primarily under a developmental control. However nitrogen fixation appears to have a quantitative effect on expression of gln- as the abundance of this mRNA is about 2 to 4-fold higher under nitrogen-fixing conditions. This effect could not be mimicked by addition of exogenous ammonium and moreover is not specific to the gln- gene as mRNA from a leghaemoglobin gene was similarly affected. Taken together these results have failed to find an effect of ammonium on specifically inducing the expression of glutamine synthetase genes in roots and nodules of P. vulgaris.     

Seedling responses to band applied NH4OH rates and to N form in two maize hybrids

Growth of maize seedlings can be improved by enhanced ammonium nutrition, but placing fertilizer anhydrous ammonia close to seedlings introduces the risk of ammonia toxicity. In this study, growth and root elongation response to rates of closely placed NH₄OH bands were investigated in two contrasting maize hybrids. Seven rates of NH₄OH, ranging from 0 to 200 mg N kg^-1 soil were injected into the center of each pot. A single rate of Ca(NO₃)₂-N was included to compare hybrids for N form preference at a moderate N rate. Three seedlings per pot were planted 5.7 cm from the injection point.Hybrid B73×LH51 produced a quadratic response in shoot growth to NH₄OH rates, whereas LH74×LH123 exhibited a significant linear decline in response to NH₄OH rate. Root length density sampled from the fertlized zone declined linearly in response to NH₄OH rate while a slight increase in root length density in unfertilized zones was observed at intermediate NH₄OH rates. Hybrids did not differ in root length density in either zone.The hybrid with greater tolerance of NH₄OH rates (B73×LH51) also showed a preference in shoot growth for NH₄-over NO₃-N at 66.7 mg N kg^-1 compared to LH74×LH123. On average across hybrids, nitrate concentrations of xylem exudate collected from detopped plants were 14.5 mmol g^-1 for Ca(NO₃)₂ treatments and 1.5 mmol g^-1 for NH₄OH treatments, indicating that contrasting N-form nutrition resulted from fertilizer treatments. Malate concentrations were higher in the NH₄OH treatment indicating that this organic acid anion may substitute for the negative charge of nitrate during enhanced ammonium nutrition in maize.The results suggest that potentially useful genetic variation exists in maize for N form preference and for tolerance to increasing ammonical-N rates.     

Light-mediated regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechococcus sp. PCC 6301

Glutamine synthetase (GS; EC 6.3.1.2) activity from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 shows a short-term regulation by light-dark transitions. The enzyme activity declines down to 30% of the original level after 2 h of dark incubation, and can be fully reactivated within 15 min of re-illumination. The loss of activity is not due to protein degradation, but rather to a reversible change of the enzyme, as deduced from the GS-protein levels determined in dark-incubated cells using polyclonal antibodies raised against Synechococcus GS. Incubation with 3-(3-4-dichlorophenyl)-1,1-dimethylurea (DCMU) also provokes GS inactivation, indicating that an active electron flow between both photosystems is necessary to maintain GS in an active state. On the other hand, the light-mediated reactivation of GS in dark-incubated cells treated with dicyclohexyl-carbodiimide (DCCD) or carbonyl cyanide m-chlorophenylhydrazone (CCCP) indicates that neither changes in the ATP synthesis nor the lack of an electrochemical proton gradient across the thylakoid membrane are directly involved in the regulation process. The inactive form of GS is extremely labile in vitro after disruption of the cells, and is not reactivated by treatment with dithiothreitol or spinach thioredoxin m. These results, taken together with the fact that dark-promoted GS inactivation is dependent on the growth phase, seem to indicate that GS activity is not regulated by a typical redox process and that some other metabolic signal(s), probably related to the ammonium-assimilation pathway, might be involved in the regulation process. In this regard, our results indicate that glutamine is not a regulatory metabolite of Synechococcus glutamine synthetase.Abbreviations CAP chloramphenicol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - DTT dithiothreitol - GOGAT glutamate synthase - GS glutamine synthetase - PFD photon flux density This work has been financed by the Directión General de Investigación Científica y Técnica, (Grant PB88-0020) and by the Junta de Andalucía, Spain.     

Regulation of nitrate uptake by amino acids in maize cell suspension culture and intact roots

The effect of amino acids on nitrate transport was studied in Zea mays cell suspension cultures and in Zea mays excised roots. The inclusion of aspartic acid, arginine, glutamine and glycine (15mM total amino acids) in a complete cell-culture media containing 1.0 mM NO₃ ^- strongly inhibited nitrate uptake and the induction of accelerated uptake rates. The nitrate uptake rate increased sharply once solution amino acid levels fell below detection limits. Glutamine alone inhibited induction in the cell suspension culture. Maize seedlings germinated and grown for 7 days in a 15 mM mixture of amino acids also had lower nitrate uptake rates than seedlings grown in 0.5 mM Ca(NO₃)₂ or 1 mM CaCl₂. As amino acids are the end product of nitrate assimilation, the results suggest an end-product feed-back mechanism for the regulation of nitrate uptake.     

Genetic,molecular and developmental analysis of the glutamine synthetase isozymes ofDrosophila melanogaster

The glutamine synthetase isozymes ofDrosophila melanogaster offer an attractive model for the study of the molecular genetics and evolution of a small gene family encoding enzymatic isoforms that evolved to assume a variety of specific and sometimes essential biological functions. InDrosophila melanogaster two GS. isozymes have been described which exhibit different cellular localisation and are coded by a two-member gene family. The mitochondrial GS structural gene resides at the 21B region of the second chromosome, the structural gene for the cytosolic isoform at the 10B region of the X chromosome. cDNA clones corresponding to the two genes have been isolated and sequenced. Evolutionary analysis data are in accord with the hypothesis that the twoDrosophila glutamine synthetase genes are derived from a duplication event that occurred near the time of divergence between Insecta and Vertebrata. Both isoforms catalyse all reactions catalysed by other glutamine synthetases, but the different kinetic parameters and the different cellular compartmentalisation suggest strong functional specialisation. In fact, mutations of the mitochondrial GS gene produce embryo-lethal female sterility, defining a function of the gene product essential for the early stages of embryonic development. Preliminary results show strikingly distinct spatial and temporal patterns of expression of the two isoforms at later stages of development.     

Regulation of the appearance of glutamine synthetase in mustard (Sinapis alba L.) cotyledons by light,nitrate and ammonium

During transformation of mustard seedlings cotyledons from storage organs to photosynthetically competent leaves, a process which occurs during the first 4 d after sowing, total glutamine-synthetase (GS, EC 6.3.1.2) activity increases from zero to the high level usually observed in green leaves. In the present study we have used ion-exchange chromatography to separate possible isoforms of GS during the development of the cotyledons. The approach failed since we could only detect a single form of GS, presumably plastidic GS, under all circumstances tested. The technique of selective photooxidative destruction of plastids in situ was applied to solve the problem of GS localization. It was inferred from the data that the GS as detected by ion-exchange chromatography is plastidic GS.The regulatory role, if any, of light, nitrate and ammonium in the process of the appearance of GS in the developing cotyledons was investigated. The results show that nitrate and ammonium play only minor roles. Light, operating via phytochrome, is the major regulatory factor.Abbreviations c continuous - D darkness - FPLC fast protein liquid chromatography - GS glutamine synthetase (L-glutamate:ammonia ligase, ADP forming, EC 6.3.1.2) - FR far-red light (3.5 W·m^-2) - NF Norflurazon - R red light (6.8 W·m^-2, _R=0.8)) - RG9-light long-wavelength FR (10 W·m^-2, _RG9<0.01) - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system