Glutamine synthetase activity,ammonia assimilation and control of nitrate reduction in the unicellular red algaCyanidium caldarium

Addition ofl-methionine-dl-sulphoximine to cells ofCyanidium caldarium brings about a loss of glutamine synthetase activity. Concomitantly ammonia assimilation is prevented.Under physiological conditions nitrate reductase [NAD(P)H: nitrate oxidoreductase EC 1.6.6.2] is reversibly converted into an inactive enzyme upon addition of ammonia. In the presence of methionine sulphoximine, when glutamine synthetase activity is lost, nitrate reductase is no longer inactivated by ammonia. It is suggested that ammonia itself is not the actual effector of nitrate reductase inactivation.Concomitantly with the failure of nitrate reductase to undergo ammonia-inactivation, in the presence of methionine sulphoximine nitrate reduction is an uncontrolled process, thus, in media with nitrate ammonia continues to be produced and excreted into the external medium at a constant rate.Abbreviations NR Nitrate reductase - GS Glutamine synthetase - GOGAT Glutamate syntase - MSX l-methionine-dl-sulphoximine     

Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor

Cysteine synthesis from sulfide and O-acetyl-L-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. Using Lemna minor, we analyzed the effects of omission of CO(2) from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5'-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO(2) led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO(2) on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO(2), APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO(2) also recovered both enzyme activities, with OAS again influenced only APR. (35)SO(4)(2-) feeding showed that treatment in air without CO(2) severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of (35)S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of (35)S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.     

Xylem exudation is related to nitrate assimilation pathway in detopped maize seedlings: use of nitrate reductase and glutamine synthetase inhibitors as tools

The xylem exudation of detopped 7-d-old seedlings of Zea maysL. doubled when KCI was present in the root medium comparedto seedlings maintained on water. It was further enhanced whenKCI was replaced by nitrogen compounds such as nitrate, ammoniumand glutamine. The role of the nitrate assimilation pathwayon the enhancement of xylem exudation rate was investigatedusing tungstate, an inhibitor of nitrate reductase (NR) activity,and phosphinothricin or methionine sulphoximine, inhibitorsof glutamine synthetase (GS) activity. The sap levels of NO₃^–,NH₄⁺, glutamine, and asparagine was used to ascertain the invivo inhibition of both enzymes. The tungstate effects werealso checked by measuring leaf in vitro NA activity and NR proteincontent. Xylem exudation rate of detopped seedlings fed withKNO₃ decreased when the nitrate assimilation pathway was blockedeither at the NR or at GS sites. This decrease was preventedwhen urea (acting as NH₄⁺ supply) was given simultaneously withtungstate. KNO₃ does not act directly on exudation, but throughthe involvement of NH₄⁺. The involvement of glutamine was alsoshown since GS inhibition resulted in a cancellation of theenhancing effect of KNO₃ on exudation. As change of exudationrate was not linked to change in sap osmolarity, it is assumedthat the assimilation chain could modify root water conductance.The role of glutamine was discussed. Key words: Exudation, maize, nitrate, conductance, NR, GS     

Glutamine synthetase of Chlamydomonas: its role in the control of nitrate assimilation

Work is described which suggests that glutamine synthetase (GS) could play an important and direct regulatory role in the control of NO₃ assimilation by the alga. In both steady-state cells and ones disturbed physiologically by changes in light or nitrogen supply the assimilation of NO₃ appears to be limited by the activity of GS. Moreover although in normal cells NH₃ can completely inhibit NO₃ uptake, promote the deactivation of nitrate reductase (NR) and repress the synthesis of NR and nitrite reductase (NIR), these controls are relaxed in cells in which GS is deactivated by treatment with L-methionine-DL-sulfoximine (MSO). It is proposed that the reversible deactivation of GS may play an important part in the regulation of NO₃ assimilation although it is still not clear whether the enzyme itself or products of its metabolism are responsible.Abbreviations GS glutamine synthetase - GS_s glutamine synthetase, synthetase activity - GS_t glutamine synthetase, transferase activity - NR nitrate reductase - NIR nitrite reductase - GDH glutamate dehydrogenase - CHX cycloheximide - MSO L-methionine-DL-sulfoximine - FAD flavine adenine dinucleotide     

Regulation of nitrate assimilation in the cyanobacterium Phormidium laminosum

The intracellular ratio of 2-oxoglutarate to glutamine has been analyzed under nutritional conditions leading to different activity levels of nitrate-assimilating enzymes in Phormidium laminosum (Agardh) Gom. This non-N₂-fixing cyanobacterium adapted to the available nitrogen source by modifying its nitrate reductase (NR; EC 1.7.7.2), nitrite reductase (NiR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) activities. The 2-oxoglutarate/glutamine ratio was similar in cells adapted to grow with nitrate or ammonium. However, metabolic conditions that increased this ratio [i.e., nitrogen starvation or l-methionine-d,l-sulfoximine (MSX) treatment] corresponded to high activity levels of NR, NiR, GS (except in MSX-treated cells) and glutamate synthase (GOGAT; EC 1.4.7.1). By contrast, metabolic conditions that diminished this ratio (i.e., addition of ammonium to nitrate-growing cells or addition of nitrate or ammonium to nitrogen-starved cells) resulted in low activity levels. The variation in the 2-oxoglutarate/glutamine ratio preceded the changes in enzyme activities. These results suggest that changes in the 2-oxoglutarate/glutamine ratio could be the signal that triggers the adaptation of P. laminosum cells to variations in the available nitrogen source, as occurs in enterobacteria.Abbreviations Chl chlorophyll - GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - GS glutamine synthetase (EC 6.3.1.2) - MSX l-methionine-d,l-sulfoximine - NiR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.7.7.2) - TP total protein This work has been partially supported by grants from the Spanish Ministry of Education and Science (DGICYT PB88-0300 and PB92-0464) and the University of the Basque Country (042.310-EC203/94). M.I.T. was the recipient of a fellowship from the Basque Government.     

Nitrogen assimilation in plants: current status and future prospects

Nitrogen (N) is the driving force for crop yields; however, excessive N application in agriculture not only increases production cost, but also causes severe environmental problems. Therefore, comprehensively understanding the molecular mechanisms of N use efficiency (NUE) and breeding crops with higher NUE is essential to tackle these problems. NUE of crops is determined by N uptake, transport, assimilation, and remobilization. In the process of N assimilation, nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamine-2-oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase) are the major enzymes. NR and NiR mediate the initiation of inorganic N utilization, and GS/GOGAT cycle converts inorganic N to organic N, playing a vital role in N assimilation and the final NUE of crops. Besides, asparagine synthetase (ASN), glutamate dehydrogenase (GDH), and carbamoyl phosphate synthetase (CPSase) are also involved. In this review, we summarize the function and regulation of these enzymes reported in three major crops—rice, maize, and wheat, also in the model plant Arabidopsis, and we highlight their application in improving NUE of crops via manipulating N assimilation. Anticipated challenges and prospects toward fully understanding the function of N assimilation and further exploring the potential for NUE improvement are discussed.     

光质对水稻幼苗初级氮同化的影响

用滤光膜过滤蓝色或红色荧光灯,得到纯的蓝光和红光,以白光为对照,研究不同光质对水稻（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）幼苗初级氮同化的影响。结果表明：蓝光促进水稻黄化幼苗吸收ＮＯ＾－３含量,并促进ＮＲ（硝酸还原酶）的诱导。在蓝光下生长５￣７ｄ的幼苗的ＮＲ、ＮＩＲ（亚硝酸还原酶）、ＧＳ（谷氨酰胺合成酶）和ＧＯＧＡＴ（谷氨酸合酶）活性均高于白光下生长的,但第１０天以后,白光下生长的幼苗酶活性最高。与白光     

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     

光质对水稻幼苗初级氮同化的影响

Pure blue(BL) or red light (RL) were obtained by filtering blue or red fluorescent lamp light through plastic filters. With the same intensity of white light(WL) as control, the effects of light quality on the primary nitrogen assimilation of rice seedlings were studied. Irradiation for 2-6 h with BL promoted the uptake of NO-3, the induction of nitrate reductase (NR), and the increase of the NO-3 content in the etiolated seedlings.Seedlings grown under BL for 5-7 d had higher activities of NR, NIR (nitrite reductase) GOGAT (glutamate synthase) as well as GS (glutamine synthetase) than those under WL. However, for more than 10 days under BL, the levels of these enzymes became lower than those of the seedlings under WL. Compared with BL or WL, RL was less effective on the primary nitrogen assimilation.     

The effect of mineral nutrition on nitrogen assimilation by intact plants ofPisum sativum L

Biologia Plantarum - The effect of macroelements on nitrogen assimilation, level of nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) and content of nitrate...     

大麦氮敏感基因型苗期对氮饥饿的生理响应

以大田试验获得的大麦氮敏感基因型BI-45为材料,利用溶液培养方法,测定了苗期株高、根长、叶绿素含量、含氮量、谷氨酰胺合成酶和硝酸还原酶活性,以及与氮代谢相关的基因（GSI-GSl-2、GSI-3、GS2、Narl、NRT2．J、NRT2-2、NRT2-3和NRT2-4）的表达。结果表明：相对于正常供氮,氮饥饿胁迫下,BI-45根和叶中的氮素利用率提高,含氮量降低,叶绿素含量减少,根冠比增加;叶片中的谷氨酰胺合成酶活性和硝酸还原酶的活性高于根,但是,与叶中的相比,根中的谷氨酰胺合成酶活性升高及硝酸还原酶活性降低的差异性更显著;与正常供氮相比,氮饥饿处理下,根中基因傩家族,基Narl和硝酸盐转运蛋白基因NRT2家族的相对表达量皆达到显著性差异,其中GSl-I、GSl-2和NRT2-2在苗期大麦氮饥饿处理下表现尤为突出,并且在6h都有上调表达。     

Effect of applied nitrate on enzymes of ammonia assimilation in nodules ofCicer arietinum L.

Summary The relationship between N₂-fixation, nitrate reductase and various enzymes of ammonia assimilation was studied in the nodules and leaves ofC. arietinum. In the nodules of the plants growing on atmospheric nitrogen, maximum activities of glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), asparagine synthetase (AS) and aspartate aminotransferase (AAT) were recorded just prior to maximum activity of nitrogenase. In nitrate fed plants, the first major peak of GDH and AS coincided with that of nitrate reductase in the nodules. With the exception of AS, application of nitrate decreased the activities of all these enzymes in nodules but not in leaves. Activities of GS, GOGAT and AAT were affected to much greater extent than that of GDH. On comparing the plants grown without nitrate and those with nitrate, the ratios of the activities of GDH/GS and GDH/GOGAT in nitrate given plants, increased by 4 and 12 fold, respectively. The results presented in this paper suggest that in nodules of nitrate fed plants, assimilation of ammonia via GDH assumes much greater importance.     

Impact of atmospheric CO2 on growth, photosynthesis and nitrogen metabolism in cucumber (Cucumis sativus L.) plants

Expression and activity of nitrate reductase (NR; EC 1.6.6.1) and glutamine synthetase (GS; EC 6.3.1.2) were analysed in relation to the rate of CO(2) assimilation in cucumber (Cucumis sativus L.) leaves. Intact plants were exposed to different atmospheric CO(2) concentrations (100, 400 and 1200microLL(-1)) for 14 days. A correlation between the in vivo rates of net CO(2) assimilation and the atmospheric CO(2) concentrations was observed. Transpiration rate and stomatal conductance remained unaffected by CO(2) levels. The exposure of the cucumber plants to rising CO(2) concentrations led to a concomitant increase in the contents of starch and soluble sugars, and a decrease in the nitrate content in leaves. At very low CO(2), NR and GS expression decreased, in spite of high nitrate contents, whereas at normal and elevated CO(2) expression and activity were high although the nitrate content was very low. Thus, in cucumber, NR and GS expression appear to be dominated by sugar levels, rather than by nitrate contents.     

Evidence for a common genetic regulation of glutamine synthetase and nitrate uptake and reductase in the cyanobacterium Anabaena cycadeae

Summary Nitrate uptake and reductase activities of the cyanobacterium Anabaena cycadeae and its mutant, lacking glutamine synthetase, (the glutamine auxotroph) were measured. The levels of both these enzymes were up to 25-fold higher in the mutant than in the parent (Anabaena cycadeae). the data indicate operation of a common genetic regulatory mechanism controlling the loss of the primary ammonia assimilating enzyme, glutamine synthetase, and derepression of the nitrate uptake and reductase systems.Abbreviations Chl Chlorophyll - GS Glutamine Synthetase - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - SDS sodium dodecyl sulphate - Tricine N-tris(hydroxymethyl) methyl glycine - Tris Tris(hydroxymethyl) aminomethane     

A common transport system for methionine, L-methionine-DL-sulfoximine (MSX), and phosphinothricin (PPT) in the diazotrophic cyanobacterium Nostoc muscorum

We present evidence, for the first time, of the occurrence of a transport system common for amino acid methionine, and methionine/glutamate analogues l-methionine-dl-sulfoximine (MSX) and phosphinothricin (PPT) in cyanobacterium Nostoc muscorum. Methionine, which is toxic to cyanobacterium, enhanced its nitrogenase activity at lower concentrations. The cyanobacterium showed a biphasic pattern of methionine uptake activity that was competitively inhibited by the amino acids alanine, isoleucine, leucine, phenylalanine, proline, valine, glutamine, and asparagine. The methionine/glutamate analogue-resistant N. muscorum strains (MSX-R and PPT-R strains) also showed methionine-resistant phenotype accompanied by a drastic decrease in ³⁵S methionine uptake activity. Treatment of protein extracts from these mutant strains with MSX and PPT reduced biosynthetic glutamine synthetase (GS) activity only in vitro and not in vivo. This finding implicated that MSX- and PPT-R phenotypes may have arisen due to a defect in their MSX and PPT transport activity. The simultaneous decrease in methionine uptake activity and in vitro sensitivity toward MSX and PPT of GS protein in MSX- and PPT-R strains indicated that methionine, MSX, and PPT have a common transport system that is shared by other amino acids as well in N. muscorum. Such information can become useful for isolation of methionine-producing cyanobacterial strains.     

小麦开花后不同器官中硝酸还原酶和谷氨酰胺合成酶的活性比较

小麦开花后各器官的硝酸还原酶和谷氨酰胺合成酶均具有一定的活性,旗叶中硝酸还原酶和谷氨酰胺合成酶活性最高。开花后旗叶和根系硝酸还原酶和谷氨酰胺合成酶活性逐渐降低,颗壳和籽粒中硝酸还原酶和谷氨酰胺合成酶活性先升高,达到最大值后又降低。     

Isolation and characterisation of nitrate reductase mutants and regulation of nitrate reductase and nitrogenase in the cyanobacterium Nostoc muscorum

Summary Chlorate resistant mutants of the cyanobacterium Nostoc muscorum isolated after N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis were found to be defective/blocked in nitrate reductase (NR).The parent strain possessed active NR in the presence of nitrogen as nitrate and only basal levels of activity in ammonia and N-free grown cultures. Addition of ammonia suppressed the NR activity in the parent strain whereas addition of L-methionine DL-sulphoximine (MSX) restored NR activity. A similar repression by ammonia, glutamine and derepression with MSX were also observed for nitrogenase synthesis.One class of mutants lacked NR activity (nar ^-) whereas the specific activity of NR was low in another class of mutants (nar ^def). Unlike the parent, the mutants synthesized nitrogenase and differentiated heterocysts in the presence of nitrate nitrogen. Uptake studies of nitrite and ammonia in mutants revealed that they possessed both nitrite reductase and glutamine synthetases (GS) at low levels, and the same level respectively in comparison with the parent.     

GLUTAMINE SYNTHETASE ASE ACTYVITY IN IN CHAETOCEROS AFFINIS (BACILLARIOPHYCEAE): COMPARISON WITH OTHER ESTIMATES OF NITROGEN UTILIZATION DURING NUTRIENT PERTURBATION1

Glutamine synthftase (GS) activity was investigated in a nitratt limited continuous culture of the marine diatom Chaeloccros afTinis (Lauder) Hustedt before and after the perturbation of the culture medium with 10 μM of ¹⁵ N labelled nitrate. Parallel studies were carried out on nitrate reductase(NR). nitrate uptake and assimilation, and Ievels of cellular nitrogen containing compounds with the objective to determine the validity of the GS assay as a measure of nitrate utilization. Activities in N-deficient cells, grown at steady state, correlated well with uptake and assimilation rates. In N-sufftcient celts, however, during the nitrate pertirbation period, they accounted only for about 10% of the two latter rates, when ambient nitrate concentrations were high (0. 7-10 μ). It is proposed that under these growth conditions an alternative pathway via glutamate dehydrogenase (GDH) was operative. At low ambient nitrate concentrations (0.1-0.7 μM), GS activities, uptake and assimilation rates again balanced rather well. Thus, the data support the view that GDH activity is associated with high levels and GS with low levels of external or internal nitrogen.