Determining potential glutamine synthetase and glutamate dehydrogenase activity in soil

Ammonium (NH₄⁺), an important nitrogen (N) source for microorganisms, is assimilated via two major pathways. One route is catalyzed by glutamate dehydrogenase (GDH), while the other mechanism involves two enzymes, glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS/GOGAT enzyme system requires more energy to operate, but has a much higher affinity for NH₄⁺ than GDH. We describe procedures to determine potential GS and GDH activity in soil samples. GS and GDH are intracellular enzymes. We used chloroform fumigation to make cell membranes permeable for substrates and products of the enzymes. Fumigation for 4 h increased GS activity almost ten-fold compared to the unfumigated control. Under optimized assay conditions, GS activity increased linearly for at least 80 min, indicating that the substrates were not limiting. In contrast to what was found for GS activity, direct addition of substrates to the soil to assay GDH activity did not result in a linear increase in GDH activity over time. A linear response for 3 h, however, resulted when the soil samples were first extracted with buffer solution and the reagents were added after centrifugation. The differences between the assays explain why fumigation for 3 d prior to the assay increased GDH activity by only 60%. In a microcosm study with glucose and NH₄⁺ addition, the activity of the two enzymes depended on the carbon (C) to N ratio of the amendment. With increasing C to N ratios from 5 to 120, GS activity doubled, while C to N ratios higher than 120 did not further increase GS activity. In contrast, GDH activity decreased by 13% with increasing C to N ratios from 5 to 200. The GDH to GS activity ratio in soil may therefore yield valuable information about the availability of N relative to C at a specific time.     

毛白杨对15N-硝态氮和铵态氮的吸收、利用及分配

以毛白杨新无性系50号插条苗为试材,应用15N示踪技术研究在相同施氮量下毛白杨(Populus tomentosa)苗木对不同形态氮素的吸收、分配及利用特性。结果表明：不同处理下毛白杨苗木在施肥1周后对肥料氮的吸收呈逐渐上升趋势,并在施肥后28d达到最大值,NO3-15N处理苗木全氮量为0.67g/株, NH4-15N处理苗木全氮量为0.60g/株;吸收NO3-15N为0.26g/株,吸收NH4-15N为0.12g/株,分别占苗木全氮的比例39.15%和19.95%。毛白杨苗木对两种氮素的利用程度差异显著,在利用率最高时期,NO3-15N利用率可达25.83%,约为NH4-15N（12.03%）的2倍。氮素在各器官中分配差异显著,总体趋势为叶﹥根﹥茎。叶中NO3-15N的分配率显著高于NH4-15N     

Cytosolic glutamine synthetase is present in the phloem sap of rice (Oryza sativa L.)

The presence of glutamine synthetase (GS) in the rice sieve tube was examined. Proteins in the rice phloem sap from leaf sheaths were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes and immunoblotted with anti-GS1 antibody. A cross-reacting band, thought to be GS1, was detected in the phloem sap. Moreover, the phloem sap contained a significant amount of GS transferase activity. Previous studies have shown that the concentrations of substrates and cofactors in the rice phloem sap are sufficient for cytosolic GS reaction. These data suggest that physiologically active GS1 is present in rice phloem sap, which might convert glutamate to glutamine in vivo .     

Ammonium (15N) metabolism in cotton under salt stress

Plant growth and metabolism is impaired under stress conditions, resulting in decreased crop yields. The purpose of this investigation was to evaluate the NaCl stress effects on NH⁺ ₄ metabolism in cotton plants at vegetative and reproductive stages of growth.

Cotton (Gossypium hirsutum L.) plants grown in normal (control) and NaCl treated Hoagland solutions were analyzed for distribution of N¹⁵ in NH⁺ ₄ plus amide‐N, free α‐amino‐N, total soluble‐N and protein‐N after the plants were provided ¹⁵NH₄NO₃ in nutrient solutions for 6, 12 and 24 h. The concentration of protein‐¹⁵N was enhanced under a low level of NaCl (‐0.4 MPa osmotic potential) at the vegetative growth stage. The difference between the protein‐¹⁵N concentration of the moderately salinized (‐0.8 MPa) plants and the controls was not significant. A high level of NaCl (‐1.2 MPa) significantly decreased protein‐N content of plants compared with the controls and any other level of salinity. The NaCl increased accumulation of NH₄ ⁺ plus amide‐N, free (α‐amino‐N, and total soluble‐N in cotton shoots, at both stages of growth. Low osmotic potential (high osmotic pressure) of the nutrient solution induced by excessive amounts of NaCl in nutrient solution inhibited NH⁺ ₄ metabolism and decreased protein synthesis, thus resulting in accumulation of soluble N‐compounds. The ionic effect probably contributed also to inhibition of protein synthesis.     

Preparation of Ammonium‐15N and Nitrate‐15N Samples by Microdiffusion for Isotope Ratio Analysis by Optical Emission Spectrometry

Abstract

A diffusion method for the preparation and measurement of ¹⁵N abundance of ammonium and nitrate in KCl extracts of soil using optical emission spectrometry (OES) was compared with conventional continuous flow isotope ratio mass spectrometry (IRMS). There were highly significant correlations between the values obtained by using OES and IRMS. The 99% confidence interval of the intercept included the value 0 and the 99% confidence interval of the slope included the value 1 for both nitrate and ammonium measurements, suggesting that the results from the two methods did not differ significantly. In another experiment, ¹⁵N values of nitrate and ammonium from soil extracts prepared by using the standard distillation procedures for OES were compared against the microdiffusion preparation method. Again, there was a highly significant correlation between the values: the 95% confidence interval of the intercept included the value 0, and the 95% confidence interval of the slope included the value 1, again suggesting that the two methods did not differ significantly. It was concluded that the diffusion technique is an appropriate and simple method of sample preparation for inorganic N analysis of KCl extracts using OES.     

Some regulative properties of glutamine synthetase in cucumber leaves

Glutamine synthetase (L-glutamate: ammonia ligase (ADP) EC 6. 3. t 2) was prepared from cucumber leaves grown on ammonium medium and some regulative properties were investigated. The apparent Michaelis constant (Km) values of the various substrates and cofactors were determined. Lineweaver-Burk plots gave a Km of 4.5mM with L-glutamate, 0.74mM with ATP and 3.1 mM with NH₂OH. Metals, including Cu²⁺, Hg²⁺, Zn²⁺, Ni²⁺, Fe²⁺, and Fe³⁺, strongly inhibited the enzyme activity, Cd²⁺ did not inhibit the enzyme activity markedly, in contrast with its effect on the rice enzyme. Ca²⁺ was quite inhibitive to enzyme reaction and more than 50% of the activity was lost at 3 mM. The amino acids tested generally had no effect on the enzyme activity except alanine, which showed little but clear inhibition. Isocitrate and α-ketoglutarate were slightly promotive to enzyme activity while pyruvate and glyoxylate (24 mM) significantly inhibited the enzyme activity. Glucose-l- or -6-phosphate and fructose-6-phosphate were inhibitive to similar degrees, about 20% at 22.5 mM, and 3-phosphoglycerate (22.5 mM) markedly inhibited the enzyme activity up to 56%. Among the nucleotides tested, UTP, CTP, and GTP inhibited slightly, and marked inhibitions of H and 78% were observed after the addition of AMP (5 mM) and ADP (5 mM) respectively. Pyridoxal-5′-phosphate, which is a characteristic incompetitive inhibitor of L-glutamate and NH₂-OH, here inhibited enzyme activity significantly. On the other hand, pyridoxamine-5′-phosphate had almost no effect. The inhibition caused by the former was not recovered by the latter. This response of glutamine synthetase to both compounds was in agreement with the idea that the nitrogen status of the plant could be reflected by the ratio pyridoxamine phosphate/pyridoxal phosphate. Also this suggested that the regulative properties of glutamine synthetase with respect to pyridoxal or pyridoxamine phosphate could be understood from the point of view of the economical use of nitrogen.     

Resistance of Medicago truncatula to salt stress is related to glutamine synthetase activity and sodium sequestration

The response to salinity may largely vary not only among species but even cultivars or lines of the same species. Knowledge of the reasons underlying these differential responses can be critical in breeding programs to obtain lines with enhanced performance under salinity. In this work, the responses to salt stress of three Medicago truncatula lines with contrasting salt resistance, TN6.18 (sensitive), Jemalong (reference line), and TN8.20 (resistant), have been assessed by analyzing a full array of nodule parameters (water relations, carbohydrates, ion concentrations, and enzyme activities). The aim of this work was to look for the most important criteria conferring resistance to the M. truncatula‐Sinorhizobium symbiosis under salinity. The resistance of M. truncatula to salt stress was related to nodule osmotic adjustment due to both sequestration of sodium and accumulation of soluble carbohydrates and free amino acids following protein degradation, together with an adequate nitrogen metabolism due to maintaining relatively high glutamine synthetase activity (GS) and stimulation of NADH‐dependent glutamate dehydrogenase (GDH). Glutamine synthetase activity differed clearly between the three studied lines. Thus, it may have a key role in the resistance of Medicago truncatula to salt stress.     

Natural Nitrogen-15 Abundance of Ammonium Nitrogen and Fixed Ammonium in Soils

The present article deals with the natural nitrogen-15 abundance of ammonium nitrogen and fixed ammonium in different soils.Variations in the natural ^15N abundance of ammonium nitrogen mineralized in soils under anaerobic incubation condition were related to soil pH.The δ ^15N of mineralizable N in acid soils was lower but that in neutral and calcareous soils was higher compared with the δ ^15N of total N in the soils.A variation tendence was also found in the δ ^15N of amino-acid N in the hydrolysates of soils.The natural ^15N abundance of fixed ammonium was higher than that of total N in most surface soils and other soil horizons,indicating that the increase of δ ^15N in the soil borizons beneath subsurface horizon of some forest soils and acid paddy soils was related to the higher δ ^15N value of fixed ammonium in the soil.     

Uptake of 15N by wetland rice in response to application of 15N-labelled Sesbania rostrata and urea

 Pot experiments were carried out to evaluate the response of rice to Sesbania rostrata green manure N as compared to urea fertilizer N under flooded conditions. After growing S. rostrata for 21 days with a ¹⁵N-labelled N source, the labelled Sesbania was applied to wetland rice as a green manure and the uptake of ¹⁵N from this substrate was compared to that from labelled urea. Rice was cultivated twice in the same pots. The rice was grown for a period of 49 days in each case, separated by a period of 21 days when the soil was allowed to dry. The ¹⁵N content of the soil and shoots and roots of rice was determined and ¹⁵N balances established. The total N content of the shoots and roots of rice was determined by a non-tracer method. The percentage recovery of ¹⁵N from shoot material which was derived from urea N was more than twice that from S. rostrata. The recovery of ¹⁵N from the pots receiving both green manure and urea was low, and not significantly different from that recovered from the green manure treatment. As much as 64.5–73.5% and 40.1–41% of the ¹⁵N remained in the soil which had received green manure or urea, respectively. The overall recoveries of ¹⁵N varied between 86.5% and 94.4%. At the second harvest, the oven-dry weight of shoots was significantly (P<0.05) higher in green-manure treated pots, but the total N content did not differ significantly. Labelled N remaining in the soil after amendment with the green manure was much more available to the rice crop than that remaining after the addition of urea-N. The total recovery of labelled N (shoots plus roots) amounted to 65.5% and 74%, respectively of the residual labelled N in the two S. rostrata treatments (i.e. 19.55 mg ¹⁵N pot^–1 and 39.10 mg ¹⁵N pot^–1) and 23.2% and 23.2% of the residual labelled N in the two urea treatments (i.e. 19.55 mg ¹⁵N pot and 39.10 mg ¹⁵N pot^–1), respectively. Received: 8 December 1997     

氮肥对水稻氮素吸收及利用效率的影响

选用超级稻品种沈农265和传统品种辽粳294为试材,比较氮肥对两品种氮素吸收及利用效率的影响。结果表明,氮肥施用量高时,沈农265氮素农学效率、生理效率等指标高于辽粳294,氮肥用量低时则相反,说明高氮肥条件有利于沈农265潜力的发挥;同样肥力水平下,沈农265每100 kg子粒所需氮量少,说明其氮素向子粒转化、运输能力强于辽粳294。     

标记停留时间对水稻及其生物质炭的13C和15N丰度的影响

稳定同位素技术是研究土壤元素循环的重要技术手段。本实验研究了同位素标记后的停留时间对水稻地上和地下部分及利用其制备的生物质炭的13C丰度（δ13C）和15N丰度（δ15N）的影响，为深入研究生物质炭对土壤碳、氮过程的影响提供基础。研究以水稻为材料，利用15N-尿素叶面喷施和13C-CO2脉冲标记的方法对水稻进行了15N和13C双标记，15N标记结束后设置4 h、6 h和24 h三个停留时间，将标记后的水稻分为地上和地下部分，分别在300 °C和500 °C下制备成生物质炭，利用同位素质谱仪测定水稻及其生物质炭的δ13C和δ15N。结果表明，随着停留时间的延长，水稻地上部分的δ13C由872‰逐渐降低至578‰，而地下部分的δ13C由226‰逐渐升高至869‰。与δ13C不同，水稻地上部分δ15N呈现先增加后降低的趋势，停留时间6 h时δ15N最大（1764‰），而地下部分的δ15N呈现先降低后增加的趋势。整体而言，与水稻原料相比，生物质炭的δ13C和δ15N分别降低了52.1%和15.9%。而且，生物质炭的δ13C和δ15N均在停留时间为24 h时最高，300 °C生物质炭表现的更加明显。随着停留时间的延长，300 °C生物质炭的热水可提取有机碳的δ13C比残留固体的δ13C降低的比例由4.14%提高到11.0%，而对于500 °C生物质炭则由32.3%降低到18.9%，表明延长停留时间分别降低和提高了300 °C和500 °C生物质炭的13C均匀性。综上所述，本研究发现标记后的停留时间对水稻δ13C和δ15N的影响不同，并且这种影响没有延续至生物质炭，停留时间和制备温度共同影响水稻生物质炭的13C均匀性。     

Ammonium uptake capacity and response of cytosolic glutamine synthetase 1;2 to ammonium supply are key factors for the adaptation of ammonium nutrition in Arabidopsis thaliana

Plant requires nitrogen for the growth, and it use nitrate and ammonium from the environment. Plant suffers from the toxicity when excess ammonium is supplied as a sole nitrogen, although it could be a good nitrogen source for plant growth. We hypothesized that the different responses of ecotypes to ammonium nutrient could partly account for the adaptation of Arabidopsis to an ammonium environment. The purpose of this study is to understand the different responses of ecotypes in ammonium environment. The growth of Arabidopsis thaliana ecotypes, Columbia was compared to those of Arabidopsis thaliana ecotypes, Landsberg erecta in ammonium nutrient. The ratio of shoot dry weight to root dry weight was compared to evaluate the adaptation of two ecotypes. The shoot:root ratio of Landsberg was significantly higher than that of Columbia. T-DNA insertion in cytosolic glutamine synthetase 1;2, one of the essential ammonium assimilatory enzymes, led a decrease of shoot:root ratio. We also measured the isotope-labeled ammonium uptake and the expression levels of ammonium transporter genes, and also the expression of ammonium assimilatory genes, glutamine synthetase genes and glutamate synthase genes, in roots after ammonium re-supply using real-time polymerase chain reaction analysis. We found that (1) ammonium uptake of Landsberg erecta was higher than that of Columbia, when ammonium was supplied at higher concentration, and (2) cytosolic glutamine synthetase 1;2 was highly increased by ammonium supply in the root of Landsberg erecta. The present study suggested the importance of these two factors for adaptation of Arabidopsis to an ammonium-rich environment.     

氮肥管理对 15N标记水稻秸秆氮吸收利用的影响

利用15N同位素示踪技术,通过盆栽模拟试验揭示氮肥管理方式对水稻秸秆氮吸收利用的影响,设置基肥氮素在插秧前施入和基肥氮素与秸秆同时施入(调节C/N)两个处理,试验结果表明,秸秆中的氮素可以作为下茬作物生长的有效氮源;基肥氮素与秸秆同时施入(调节C/N)处理秸秆氮的利用率显著高于基肥氮素在插秧前施入,水稻从~(15)N秸秆标记物中摄取氮的比例(Ndf_S)明显低于土壤肥料中氮(Ndf_(S+F));水稻地上植株对秸秆氮的综合利用率为6.51%～7.99%,其中,茎叶秸秆氮利用率为1.40%～1.75%,穗利用率为5.11%～5.25%。在秸秆还田的条件下,基肥氮素与秸秆同时施入,可以显著提高水稻对秸秆氮素的吸收能力。     

Recovery of 15N Labelled Urea as Affected by Fixation of Ammonium by Clay Minerals

A greenhouse experiment was conducted with Lolium perenne to determine whether ammonium (NH₄⁺-N) fixation by clay minerals can increase the recovery of nitrogen following application of ¹⁵N labelled urea. A silty loam subsoil, B_t horizon from an Alfisol derived from loess, pH (CaCl₂) 7.9, was chosen for the experiment. The NH₄⁺-N fixation capacity was altered by varying the distribution of potassium (K) in the upper and lower soil layer. In the K₀ treatment (control), the upper soil layer fertilized with urea was not supplied with K, whereas the lower soil layer was fertilized with 300 mg K kg^?1 soil. In the K₁^?, K₂^? and K₃ treatment the upper soil was supplied with 100-, 200- and 300 mg K kg^?1 soil, respectively. The soil in the lower layer of the K₃ treatment was not supplied with K. The recovery of ¹⁵N from applied urea (¹⁵N uptake of Lolium perenne plus residual soil ¹⁵N_t) was 86.1% in the K₀ treatment and 75.2%, 69.1% and 69.6% in K_1-, K_2- and K₃ treatments, respectively, showing that ¹⁵N losses were smallest in the K₀ treatment. Two weeks after applying ¹⁵N labelled urea the amounts of nonexchangeable ¹⁵NH₄⁺-N in the upper soil layer were significantly higher in the K₀ treatment than in the treatments with K application. Apparently, NH₄⁺-N fixation by clay minerals can reduce NH₃ volatilization after urea fertilization, if the amount of exchangeable K is low.     

Carbon assimilation and microbial activity in soil

In order to characterise the term microbial ?activity”? three different microbial populations belonging to a luvisol (I), a phaeozem (II) and a rendzina (III) were used for studying kinetic parameters such as substrate affinity, growth rate, yield and turnover time and the metabolic quotient of basal respiration. Glucose was used as a carbon source. Specific growth rate values (μ) varied between 0.0037 and 0.015 h^?1 depending on soil type and glucose concentration and were far below the potential μ_max. The calculated turnover time was 3–11 days, respectively. The yield coefficient was in the range between 0.37 and 0.53. The maximal uptake rate of glucose–C of soil population (II) was 0.041 g C g^?1 biomass-C h^?1. The determined affinity constant (K_m) was 57 μg C g^?1 soil. The affinity to glucose was higher for the glucose-mediated CO₂ evolution with K_m values of 15.2 and 17.5 than for the glucose uptake system itself. The observed qCO₂ values of the basal respiration at temperature increments from 0 to 45° C were almost identical for the soils (I) and (II). The calulated Q₁₀ lay in the range between 1.4 and 2.0.     

施肥模式对中国稻田氮素径流损失和产量影响的Meta分析

优化施肥模式是减少稻田氮素径流损失,降低农业面源污染风险,确保作物产量的重要措施。采用Meta分析方法研究不同施肥模式(施肥类型、施肥量和分施次数)对稻田氮素径流损失和作物产量的影响,筛选了29个同行评议研究论文,共109组有效的配对试验数据进行分析。结果表明,与不施肥相比,化肥、有机肥替代和缓释肥替代分别增加氮素径流损失量N 6.73、1.21和3.40 kg/hm²。与单施化肥相比,有机肥和缓释肥替代分别显著降低82.0%和49.5%的氮素径流损失,而对作物产量没有显著影响。同时,单施化肥处理的氮素径流损失率(4.34%)显著高于有机肥替代(2.33%)和缓释肥替代(2.04%)。施肥量的影响表现为,当施肥量低于N 240     

磷素水平对不同大豆品种叶片及子粒谷氨酰胺合成酶活性的影响

盆栽试验选用东农42（高蛋白品种）、合丰25（中间型品种）、东农46（高油品种）3个基因型大豆品种,在每千克土壤施N和K2O各为0.033g基础上,设P0、P5、P10、P15 4个P处理（即每kg土壤分别施P2O5 0、0.033、0.067、0.100g）,研究了不同磷素水平对大豆生育期功能叶片及子粒谷氨酰胺合成酶（GS）活性的影响。结果表明,东农42和合丰25均为P10处理功能叶片和子粒GS活性最高;东农46为P10处理功能叶片GS活性最高,P5处理子粒GS活性最高。同一处理不同品种间东农42功能叶片和子粒GS活性最高,东农46最低,合丰25处于二者之间;3个品种不同处理功能叶片GS活性最高值均出现在结荚期,东农42子粒GS活性处于高值时间较长,东农46最短,合丰25处于二者之间。施磷对3个大豆品种叶片及子粒GS活性有影响,适宜的施磷量有利于提高其活性。     

Stem labeling results in different patterns of 14C rhizorespiration and 15N distribution in plants compared to natural assimilation pathways

To investigate C and N rhizodeposition, plants can be ¹³C‐¹⁵N double‐labeled with glucose and urea using a stem‐feeding method (wick method). However, it is unclear how the ¹³C applied as glucose is released into the soil as rhizorespiration in comparison with the ¹³C applied as CO₂ using a natural uptake pathway. In the present study, we therefore compared the short‐term fate of ¹⁴C and ¹⁵N in white lupine and pea plants applied either by the wick method or the natural pathways of C and N assimilation. Plants were pulse‐labeled in ¹⁴CO₂‐enriched atmosphere and ¹⁵N urea was applied to the roots (atmosphere–soil) following the natural assimilation pathways, or plants were simultaneously labeled with ¹⁴C and ¹⁵N by applying a ¹⁴C glucose–¹⁵N urea solution into the stem using the wick method. Plant development, soil microbial biomass, total rhizorespiration, and distribution of N in plants were not affected by the labeling method used but by plant species. However, the ¹⁵N : N ratio in plant parts was significantly (p < 0.05) affected by the labeling method, indicating more homogeneous ¹⁵N enrichment of plants labeled via root uptake. After ¹⁴CO₂ atmosphere labeling of plants, the cumulated ¹⁴CO₂ release from roots and soil showed the common saturation dynamics. In contrast, after ¹⁴C‐glucose labeling by the wick method, the cumulated ¹⁴CO₂ release increased linearly. These results show that ¹⁴C applied as glucose using the wick method is not rapidly transferred to the roots as compared to a short‐term ¹⁴CO₂ pulse. This is partly due to a slower ¹⁴C uptake and partly due to slow distribution within the plant. Consequently, ¹⁴C‐glucose application by the wick method is no pulse‐labeling approach. However, the advantages of the wick method for ¹³C‐¹⁵N double labeling for estimating rhizodeposition especially under field conditions requires further methodological research.