Short term physiological implications of NBPT application on the N metabolism of Pisum sativum and Spinacea oleracea

The application of urease inhibitors in conjunction with urea fertilizers as a means of reducing N loss due to ammonia volatilization requires an in-depth study of the physiological effects of these inhibitors on plants. The aim of this study was to determine how the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) affects N metabolism in pea and spinach. Plants were cultivated in pure hydroponic culture with urea as the sole N source. After 2 weeks of growth for pea, and 3 weeks for spinach, half of the plants received NBPT in their nutrient solution. Urease activity, urea and ammonium content, free amino acid composition and soluble protein were determined in leaves and roots at days 0, 1, 2, 4, 7 and 9, and the NBPT content in these tissues was determined 48 h after inhibitor application. The results suggest that the effects of NBPT on spinach and pea urease activity differ, with pea being most affected by this treatment, and that the NBPT absorbed by the plant caused a clear inhibition of the urease activity in pea leaf and roots. The high urea concentration observed in leaves was associated with the development of necrotic leaf margins, and was further evidence of NBPT inhibition in these plants. A decrease in the ammonium content in roots, where N assimilation mainly takes place, was also observed. Consequently, total amino acid contents were drastically reduced upon NBPT treatment, indicating a strong alteration of the N metabolism. Furthermore, the amino acid profile showed that amidic amino acids were major components of the reduced pool of amino acids. In contrast, NBPT was absorbed to a much lesser degree by spinach plants than pea plants (35% less) and did not produce a clear inhibition of urease activity in this species.     

新型磷酰胺类脲酶抑制剂对不同质地土壤尿素转化的影响

施用脲酶抑制剂是降低尿素水解、减少氨气挥发损失、提高作物氮(N)肥利用率的重要途径之一.采用室内恒温、恒湿模拟试验方法,在25 ℃黑暗条件下培养,研究新型磷酰胺类脲酶抑制剂N-丙基磷酰三胺(NPPT)的脲酶抑制效果,比较其与N-丁基磷酰三胺(NBPT)在不同尿素用量条件下不同质地土壤中对脲酶的抑制差异.结果表明: 在壤土和黏土中,尿素作用时间≤9 d,添加抑制剂可以将尿素水解时间延长3 d以上.砂土中,尿素分解过程相对缓慢,添加抑制剂显著降低土壤脲酶活性,抑制NH₄⁺-N生成.在培养期间,不同尿素用量条件下,脲酶抑制剂在不同质地土壤中的抑制效果表现为高施N量优于低施N量.培养第6天,在尿素用量250 mg N·kg^-1条件下,NBPT和NPPT在砂土中脲酶抑制率分别为56.3%和53.0%,在壤土中分别为0.04%和0.3%,在黏土中分别为4.1%和6.2%;尿素用量500 mg N·kg^-1,NBPT和NPPT在砂土中脲酶抑制率分别为59.4%和65.8%,在壤土中分别为14.5%和15.1%,在黏土中分别为49.1%和48.1%.不同质地土壤中脲酶抑制效果表现为砂土>黏土>壤土.不同抑制剂处理在培养期间土壤NH₄⁺-N含量呈现先上升后下降的趋势,而NO₃^--N含量和表观硝化率均呈现逐渐上升的趋势.与单施尿素处理相比,添加脲酶抑制剂NBPT和NPPT显著增加土壤中的残留尿素态N,降低NH₄⁺-N生成.新型脲酶抑制剂NPPT在不同质地土壤中的抑制效果与NBPT相似,是一款有效的脲酶抑制剂.     

Effect of <Emphasis Type="Italic">N</Emphasis>-(<Emphasis Type="Italic">n</Emphasis>-butyl) thiophosphoric triamide on urea metabolism and the assimilation of ammonium by <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

The use of urea as an N fertilizer has increased to such an extent that it is now the most widely used fertilizer in the world. However, N losses as a result of ammonia volatilization lead to a decrease in its efficiency, therefore different methods have been developed over the years to reduce these losses. One of the most recent involves the use of urea combined with urease inhibitors, such as N-(n-butyl) thiophosphoric triamide (NBPT), in an attempt to delay the hydrolysis of urea in the soil. The aim of this study was to perform an in-depth analysis of the effect that NBPT use has on plant growth and N metabolism. Wheat plants were cultivated in a greenhouse experiment lasting 4 weeks and fertilized with urea and NBPT at different concentrations (0, 0.012, 0.062, 0.125%). Each treatment was replicated six times. A non-fertilized control was also cultivated. Several parameters related with N metabolism were analysed at the end of growth period. NBPT use was found to have visible effects, such as a transitory yellowing of the leaf tips, at the end of the first week of treatment. At a metabolic level, plants treated with the inhibitor were found to have more urea in their tissues and a lower amino acid content, lower glutamine synthetase activity, and lower urease and glutamine synthetase content at the end of the study period, whereas their urease activity seemed to have recovered by this stage.     

Inhibition of jack bean urease by N-(n-butyl) thiophosphorictriamide and N-(n-butyl) phosphorictriamide: determination of the inhibition mechanism

N-(n-butyl)thiophosphorictriamide (NBPT) and its oxygen analogue N-(n-butyl)phosphorictriamide (NBPTO) were studied as inhibitors of jack bean urease. NBPTO was obtained by spontaneous conversion of NBPT into NBPTO. The conversion under laboratory conditions was slow and did not affect NBPT studies. The mechanisms of NBPT and NBPTO inhibition were determined by analysis of the reaction progress curves in the presence of different inhibitor concentrations. The obtained plots were time-dependent and characteristic of slow-binding inhibition. The effects of different concentration of NBPT and NBPTO on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships for a one-step enzyme-inhibitor interaction, qualified as mechanism A. The inhibition constants of urease by NBPT and NBPTO were found to be 0.15 microM and 2.1 nM, respectively. The inhibition constant for NBPT was also calculated by steady-state analysis and was found to be 0.13 microM. NBPTO was found to be a very strong inhibitor of urease in contrast to NBPT.     

Inhibition of Jack Bean Urease by N-(n-butyl)thiophosphorictriamide and N-(n-butyl)phosphorictriamide: Determination of the Inhibition Mechanism

N-(n-butyl)thiophosphorictriamide (NBPT) and its oxygen analogue N-(n-butyl)phosphorictriamide (NBPTO) were studied as inhibitors of jack bean urease. NBPTO was obtained by spontaneous conversion of NBPT into NBPTO. The conversion under laboratory conditions was slow and did not affect NBPT studies. The mechanisms of NBPT and NBPTO inhibition were determined by analysis of the reaction progress curves in the presence of different inhibitor concentrations. The obtained plots were time-dependent and characteristic of slow-binding inhibition. The effects of different concentration of NBPT and NBPTO on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships for a one-step enzyme-inhibitor interaction, qualified as mechanism A. The inhibition constants of urease by NBPT and NBPTO were found to be 0.15 μM and 2.1 nM, respectively. The inhibition constant for NBPT was also calculated by steady-state analysis and was found to be 0.13 μM. NBPTO was found to be a very strong inhibitor of urease in contrast to NBPT.     

Use of urease inhibitors to reduce ammonia loss following application of urea to flooded rice fields

Ammonia (NH₃) volatilization is an important mechanism for nitrogen (N) loss from flooded rice fields following the application of urea into the floodwater. One method of reducing losses is to use a urease inhibitor that retards the hydrolysis of urea by soil urease and allows the urea to diffuse deeper into the soil. The two chemicals that have shown most promise are phenylphosphorodiamidate [PPD] and N(n-butyl)thiophosphorictriamide [NBPT], but they seldom work effectively. PPD decomposes rapidly when the pH departs from neutrality, and NBPT must be converted to the oxygen analogue for it to be effective. Our field studies in Thailand show that the activity of PPD can be prolonged, and NH₃ loss markedly reduced, by controlling the floodwater pH with the algicide terbutryn. A mixture of NBPT and PPD in the presence of terbutryn was even more effective than PPD alone. It appears that during the time when the PPD was effective, NBPT was being converted to the oxygen analogue. The combined urease inhibitor-algicide treatment reduced NH₃ loss from 10 to 0.4 kg N ha^-1.     

Molecular Modeling and docking of Wheat Hydroquinone Glucosyl transferase by using Hydroquinone,Phenyl phosphorodiamate and n-(n butyl) Phosphorothiocic Triamide as Inhibitors

In agriculture high urease activity during urea fertilization causes substantial environmental and economical problems by releasing abnormally large amount of ammonia into the atmosphere which leads to plant damage as well as ammonia toxicity. All over the world, urea is the most widely applied nitrogen fertilizer. Due to the action of enzyme urease; urea nitrogen is lost as volatile ammonia. For efficient use of nitrogen fertilizer, urease inhibitor along with the urea fertilizer is one of the best promising strategies. Urease inhibitors also provide an insight in understanding the mechanism of enzyme catalyzed reaction, the role of various amino acids in catalytic activity present at the active site of enzyme and the importance of nickel to this metallo enzyme. By keeping it in view, the present study was designed to dock three urease inhibitors namely Hydroquinone (HQ), Phenyl Phosphorodiamate (PPD) and N-(n-butyl) Phosphorothiocic triamide (NBPT) against Hydroquinone glucosyltransferase using molecular docking approach. The 3D structure of Hydroquinone glucosyltransferase was predicted using homology modeling approach and quality of the structure was assured using Ramachandran plot. This study revealed important interactions among the urease inhibitors and Hydroquinone glucosyltransferase. Thus, it can be inferred that these inhibitors may serve as future anti toxic constituent against plant toxins.     

施用缓/控释尿素对玉米苗期土壤生物学活性的影响

采用盆栽试验,模拟田间生态环境,研究了施用不同种缓/控释氮素底肥对玉米苗期土壤硝酸还原酶、脲酶活性及微生物量碳、氮的影响.结果表明,施用硝化抑制剂(双氰胺)和脲酶抑制剂(n-丁基硫代磷酰三胺)涂层大颗粒尿素肥料的土壤硝酸还原酶活性最高;施用大颗粒尿素,脲酶活性最强,微生物量碳、氮最高.施用醋酸酯淀粉包膜大颗粒尿素、包膜双氰胺涂层大颗粒尿素、丙烯酸树脂包膜双氰胺涂层大颗粒尿素与不施氮肥土壤脲酶活性较高;每种处理微生物量碳与氮变化完全一致.施用醋酸酯淀粉包膜硝化和脲酶抑制剂涂层大颗粒尿素肥料,土壤微生物量碳、氮最低.同种膜材料包膜抑制剂涂层大颗粒尿素制成的缓/控释氮肥,对土壤生物学活性的影响效果好于直接包膜大颗粒尿素;丙烯酸树脂包膜大颗粒尿素制成的缓/控释氮肥,对氮素的控释效果明显好于醋酸酯淀粉包膜.     

脲酶／硝化抑制剂对土壤有效态氮、微生物量氮和小麦氮吸收的影响

研究了脲酶抑制剂(NBPT)、硝化抑制剂(DCD)及二者组合在草甸棕壤上施用对尿素态N转化及土壤总有效态N、微生物量N的影响．结果表明,尿素配施NBPT、DCD及抑制剂组合能够增加尿素水解后土壤NH4^+含量2％-53％。显著降低了氧化态N的浓度,抑制了土壤中铵态N的氧化,增加土壤总有效N34％-44％,小麦吸N量增加0．26％-6．79％。其中以脲酶抑制剂与硝化抑制剂组合的效果最明显．抑制剂施用增加了微生物在小麦生长初期对有效态N固持,有利于后期土壤有效态N的矿化．     

生物炭配施硝化/脲酶抑制剂对亚热带水稻土活性氮气体排放的影响

探究施用生物炭和脲酶抑制剂/硝化抑制剂对亚热带水稻土氮素硝化过程的调控作用、氨挥发和N₂O排放的温室效应潜能的影响,确定生物炭与硝化和脲酶抑制剂的最佳组合,可为削减施用氮肥带来的活性氮气体排放对环境的负面风险提供理论依据。本研究采用室内好气培养试验方式,以单施尿素(N)为对照,设置7个试验处理[尿素+生物炭(NB),尿素+硝化抑制剂(N+NI),尿素+脲酶抑制剂(N+UI),尿素+硝化抑制剂+脲酶抑制剂(N+NIUI),尿素+硝化抑制剂+生物炭(NB+NI),尿素+脲酶抑制剂+生物炭(NB+UI),尿素+硝化抑制剂+脲酶抑制剂+生物炭(NB+NIUI)],观测生物炭与脲酶抑制剂(NBPT)/硝化抑制剂(DMPP)配施下土壤无机氮含量、N₂O排放及氨挥发的变化动态。结果表明: 1)培养期间,与N处理(5.11 mg N·kg^-1·d^-1)相比,NB处理的土壤硝化速率常数显著增加33.9%,N+NI处理显著降低22.9%;NB处理显著提高了氨氧化细菌(AOB)丰度,增幅达56.0%。2)与N处理相比,N+NI和NB+NI处理的NH₃累积排放量均显著增加约49%;N+UI处理降低了NH₃累积损失量,NB+UI处理抑制效果更明显。3)各处理的N₂O排放速率高峰均出现在施肥后前10 d;NB处理的N₂O排放高峰出现最早,N处理排放速率最高(5.87 μg·kg^-1·h^-1);硝化抑制剂与脲酶抑制剂配施减少土壤N₂O排放的效果最佳。综合计算各处理直接N₂O和间接N₂O(NH₃)排放产生的温室效应潜能(GWP)发现,N+NI和NB+NI处理较N处理分别增加了34.8%和40.9%,而NB和NB+UI处理的GWP显著降低了45.9%和60.5%。因此,生物炭与脲酶抑制剂配施对降低土壤活性氮气体排放所产生的温室效应潜能效果最佳。     

脲酶/硝化抑制剂在黑土和褐土中对尿素氮转化的调控效果

本试验研究脲酶/硝化抑制剂不同组合在黑土和褐土中对尿素水解和硝化作用的调控效果,旨在筛选出适合东北黑土、褐土的高效抑制剂组合.采用室内恒温、恒湿培养试验,以不施氮肥(CK)和施用普通尿素肥料(U)为对照,研究分别添加脲酶抑制剂N-丁基硫代磷酰三胺(NBPT)及其与硝化抑制剂双氰胺(DCD)、3,4-二甲基吡唑磷酸盐(D...     

添加生化抑制剂和腐植酸的稳定性增效尿素在黄土中的施用效果

采用田间盆栽试验,研究生化抑制剂与生物刺激素腐植酸结合制成的高效稳定性增效尿素肥料在黄土中的氮素转化特征、增产效果和氮素肥料表观利用率,以探明其施用效果,为开发适宜黄土施用的新型增效尿素肥料提供理论依据。本研究以不施氮肥(CK)和施尿素氮肥(N)为对照,在尿素中分别添加腐植酸(F)、N-丁基硫代磷酰三胺(NBPT)、3,4-二甲基吡唑磷酸盐(DMPP)和2-氯-6-三甲基吡啶(CP),以及腐植酸与3种生化抑制剂分别组合(NBPT+F、DMPP+F、CP+F)。结果表明: 与N处理相比,F、NBPT+F、DMPP+F和CP+F处理均能显著提高玉米的产量、叶片叶绿素含量、叶面积指数和植株吸氮量,对土壤铵态氮和硝态氮含量也有显著影响。与单独施用生化抑制剂相比,添加腐植酸可提高玉米叶片叶绿素含量。与CP相比,CP+F玉米的植株吸氮量、叶绿素含量、氮肥吸收利用率均显著提高;与NBPT相比,NBPT+F硝化抑制率提高10.7%,但玉米产量、叶面积指数、植株吸氮量和氮肥利用率等均有所降低;与DMPP相比,DMPP+F显著降低了玉米产量、叶面积指数、植株吸氮量、氮肥利用率和硝化抑制率等。综合玉米产量、植株吸氮量、氮肥吸收利用率以及土壤铵态氮、硝态氮含量等指标,在黄土地区施用尿素肥料时,建议添加腐植酸和CP以提升尿素性能,从而提高产量和肥料利用率。     

Inhibition of soil urease activity by amido derivatives of phosphoric and thiophosphoric acids

Summary A laboratory experiment was conducted to study inhibition of soil urease activity by amido derivatives of phosphoric and thiophosphoric acids. Results showed that derivatives with higher amido substitutions have greater inhibitory effect on urea hydrolysis in the soils used in our study. Triamides of phosphoric and thiophosphoric acids were found to be very effective inhibitors of soil urease. These compounds seem to have potential as fertilizer amendments for inhibiting soil urease activity and for improving the efficiency of nitrogen use from urea.     

碳添加下黑钙土胞内、胞外脲酶活性变化及其机制

土壤脲酶作为能够催化尿素水解的最重要酶类,对草地生态系统氮素供应具有重要作用。目前探讨不同碳添加对草地土壤胞外脲酶影响的研究报道相对较多,但碳添加对土壤胞内脲酶的影响,以及胞内和胞外脲酶对碳添加的响应是否一致等尚需深入研究。本研究依托额尔古纳森林草原过渡带生态系统研究站开展的碳添加野外试验平台(以葡萄糖为碳源),选取无碳添加(C₀)、250(C₂₅₀)和500(C₅₀₀) kg C·hm^-2·a^-1处理为供试对象,探讨碳添加下黑钙土胞内、胞外脲酶活性响应及其与土壤性质的关系。结果表明: 碳添加显著提高了土壤胞内脲酶活性,增加了土壤胞内脲酶活性占总脲酶活性的比例,但对土壤胞外脲酶活性没有显著影响。土壤胞内脲酶活性与微生物生物量具有显著正相关关系,表明胞内脲酶活性增加主要是由微生物生物量增加引起的。结构方程模型(SEM)分析表明,碳添加通过影响土壤微生物生物量间接提高了土壤胞内脲酶活性。     

Role of glutamate dehydrogenase and phosphoenolpyruvate carboxylase activity in ammonium nutrition tolerance in roots

Spinach (Spinacea oleracea L. “Correnta F1”) and pea (Pisum sativum L. “Macrocarpon”) plants were grown in a hydroponic culture with nitrate (5 mM), or ammonium (5 mM) as the nitrogen source. Dry matter accumulation declined dramatically in spinach plants fed with ammonium, whereas there was no change in pea plants when compared with nitrate-fed plants. Data obtained from δ¹⁵N, the organic nitrogen content, N-assimilation enzyme activity, glutamine synthetase (L-glutamate:ammonia-ligase; EC 6.3.1.2), glutamate dehydrogenase (L-glutamate:NAD⁺-oxidoreductase; EC 1.4.1.2) and enzymes from the tricarboxylic acid cycle suggest that ammonium incorporation into organic nitrogen is localized in the roots in pea plants and in the shoots in spinach plants. Distribution of incorporated ammonium (in shoots and roots) may determine ammonium tolerance. Our results show that unlike in spinach plants, in pea plants, an ammonium-tolerant species, GDH enzyme plays an important role in ammonium detoxification by its incorporation into amino acids. Furthermore, phosphoenolpyruvate carboxylase (phosphate:oxaloacetate-carboxy-lyase; EC 4.1.1.31) and pyruvate kinase (ATP:pyruvate-2-O-phosphotransferase; EC 2.7.1.40) activities reflect a major flow of carbon for ammonium assimilation through oxalacetate in pea plants and through pyruvate in spinach plants. The differences in the sensitivity to ammonium between the species are discussed in terms of differences in the site of ammonium assimilation as well as in the nitrogen assimilation ways.     

高寒灌丛生长季土壤转化酶与脲酶活性对增温和植物去除的响应

对青藏高原东缘窄叶鲜卑花土壤转化酶与脲酶活性对增温(0.6～1.3 ℃)和植物去除的响应进行研究,以了解气候变暖和植被干扰对高寒灌丛生长季不同时期土壤生态过程的影响.结果表明: 增温在整个生长季节使去除/不去除植物处理土壤转化酶活性显著增加了3.7%～13.3%.增温除在生长季末期对不去除植物处理土壤脲酶活性影响不显著以外,在其他时期使去除/不去除植物处理土壤脲酶活性显著增加10.8%～56.3%.去除植物处理对土壤转化酶与脲酶活性的影响因增温与生长季节而存在显著差异.去除植物显著降低了不增温样方生长季初期和末期与增温样方整个生长季节土壤转化酶活性,而没有显著影响生长季中期不增温样方土壤转化酶活性.去除植物仅在生长季末期使不增温样方土壤脲酶活性显著降低了10.5%;而在增温样方,去除植物仅在生长季初期和中期使土壤脲酶活性显著降低16.0%～18.7%.以上结果有利于全面认识高寒灌丛生态系统土壤碳氮循环过程.     

桂西北喀斯特峰丛洼地不同植被演替阶段的土壤脲酶活性

以桂西北喀斯特峰丛洼地不同演替阶段植被群落为研究对象,采用空间代替时间序列的方法,选取立地条件基本相似的草地、乔灌林和次生林3种次生演替植被,并以原生林为对照,通过野外调查取样和室内分析,探讨植被不同演替阶段土壤脲酶活性的变化特征及其与土壤理化性质的关系。结果发现,(1)不同植被演替阶段的土壤脲酶活性存在显著差异,草地最高(0.462 mg · g^-1 · d^-1),次生林次之(0.410 mg · g^-1 · d^-1),灌木林再次(0.371 mg · g^-1 · d^-1),原生林最低(0.194 mg · g^-1 · d^-1);(2)在喀斯特区域,土壤脲酶活性与全钾、粘粒含量、容重、碳氮比(C/N)、碱解氮占全氮的比例(AN/TN)呈正相关(P<0.01),与其他指标,如有机碳、全氮、碱解氮、微生物碳、微生物氮等均呈极显著负相关(P<0.01);(3)与脲酶活性关系密切的理化性质有全氮、碱解氮、微生物量、粘粒含量及C/N、AN/TN等。并不是所有区域的土壤脲酶活性都与SOC、TN、AN、微生物量呈正相关,当土壤养分较高,即土壤中的氮量不再是作物生长的限制因子时,脲酶活性有可能与之呈负相关。     

Oxidation-reduction properties of chloroplast thioredoxins, ferredoxin:thioredoxin reductase, and thioredoxin f-regulated enzymes

Oxidation-reduction midpoint potentials were determined, as a function of pH, for the disulfide/dithiol couples of spinach and pea thioredoxins f, for spinach and Chlamydomonas reinhardtii thioredoxins m, for spinach ferredoxin:thioredoxin reductase (FTR), and for two enzymes regulated by thioredoxin f, spinach phosphoribulokinase (PRK) and the fructose-1,6-bisphosphatases (FBPase) from pea and spinach. Midpoint oxidation-reduction potential (Em) values at pH 7.0 of -290 mV for both spinach and pea thioredoxin f, -300 mV for both C. reinhardtii and spinach thioredoxin m, -320 mV for spinach FTR, -290 mV for spinach PRK, -315 mV for pea FBPase, and -330 mV for spinach FBPase were obtained. With the exception of spinach FBPase, titrations showed a single two-electron component at all pH values tested. Spinach FBPase exhibited a more complicated behavior, with a single two-electron component being observed at pH values >/= 7.0, but with two components being present at pH values <7.0. The slopes of plots of Em versus pH were close to the -60 mV/pH unit value expected for a process that involves the uptake of two protons per two electrons (i. e., the reduction of a disulfide to two fully protonated thiols) for thioredoxins f and m, for FTR, and for pea FBPase. The slope of the Em versus pH profile for PRK shows three regions, consistent with the presence of pKa values for the two regulatory cysteines in the region between pH 7.5 and 9.0.     

The crystal structure of Sporosarcina pasteurii urease in a complex with citrate provides new hints for inhibitor design

Urease, the enzyme that catalyses the hydrolysis of urea, is a virulence factor for a large number of ureolytic bacterial human pathogens. The increasing resistance of these pathogens to common antibiotics as well as the need to control urease activity to improve the yield of soil nitrogen fertilization in agricultural applications has stimulated the development of novel classes of molecules that target urease as enzyme inhibitors. We report on the crystal structure at 1.50-Å resolution of a complex formed between citrate and urease from Sporosarcina pasteurii, a widespread and highly ureolytic soil bacterium. The fit of the ligand to the active site involves stabilizing interactions, such as a carboxylate group that binds the nickel ions at the active site and several hydrogen bonds with the surrounding residues. The citrate ligand has a significantly extended structure compared with previously reported ligands co-crystallized with urease and thus represents a unique and promising scaffold for the design of new, highly active, stable, selective inhibitors.