A physiological and molecular study of the effects of nickel deficiency and phenylphosphorodiamidate (PPD) application on urea metabolism in oilseed rape (Brassica napus L.)

Background and aims

Urea is the major nitrogen (N) form supplied as fertilizer in agriculture. However, urease, a nickel-dependent enzyme, allows plants to use external or internally generated urea as a nitrogen source. Since a urease inhibitor is frequently applied in conjunction with urea fertilizer, the N-metabolism of plants may be affected. The aim of this study was to determine physiological and molecular effects of nickel deficiency and a urease inhibitor on urea uptake and assimilation in oilseed rape.

Methods

Plants were grown on hydroponic solution with urea as the sole N source under three treatments: plants treated with nickel (+Ni) as a control, without nickel (?Ni) and with nickel and phenylphosphorodiamidate (+Ni+PPD). Urea transport and assimilation were investigated.

Results

The results show that Ni-deficiency or PPD supply led to reduced growth and reduced ¹⁵N-uptake from urea. This effect was more pronounced in PPD-treated plants, which accumulated high amounts of urea and ammonium. Thus, Ni-deficiency or addition of PPD, limit the availability of N and decreased shoot and root amino acid content. The up-regulation of BnDUR3 in roots indicated that this gene is a component of the stress response to nitrogen-deficiency. A general decline of glutamine synthetase (GS) activity and activation of glutamate dehydrogenase (GDH) and increases in its expression level were observed in control plants. At the same time, in (?N) or (+Ni+PPD) treated plants, no increases in GS or GDH activities and expression level were found.

Conclusions

Overall results showed that plants require Ni as a nutrient (while most widely used nutrient solutions are devoid of Ni), whether they are grown with or without a urea supply, and that urease inhibitors may have deleterious effects at least in hydroponic grown oilseed rape.     

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.     

The role of nitrogen-efficient cultivars in sustainable agriculture

To improve nitrogen (N) efficiency in agriculture, integrated N management strategies that take into consideration improved fertilizer, soil, and crop management practices are necessary. This paper reports results of field experiments in which maize (Zea mays L.) and oilseed rape (Brassica napus L.) cultivars were compared with respect to their agronomic N efficiency (yield at a given N supply), N uptake efficiency (N accumulation at a given N supply), and N utilization efficiency (dry matter yield per unit N taken up by the plant). Under conditions of high N supply, significant differences among maize cultivars were found in shoot N uptake, soil nitrate depletion during the growing season, and the related losses of nitrate through leaching after the growing season. Experiments under conditions of reduced N supply indicated a considerable genotypic variation in reproductive yield formation of both maize and oilseed rape. High agronomic efficiency was achieved by a combination of high uptake and utilization efficiency (maize), or exclusively by high uptake efficiency (rape). N-efficient cultivars of both crops were characterized by maintenance of a relatively high N-uptake activity during the reproductive growth phase. In rape this trait was linked with leaf area and photosynthetic activity of leaves. We conclude that growing of N-efficient cultivars may serve as an important element of integrated nutrient management strategies in both low- and high-input agriculture.     

Modeling nitrogen uptake in oilseed rape cv Capitol during a growth cycle using influx kinetics of root nitrate transport systems and field experimental data

The use of kinetic equations of NO3- transport systems in oilseed rape (Brassica napus), determined by 15NO3- labeling under controlled conditions, combined with experimental field data from the INRA-Chalons rape database were used to model NO3- uptake during the plant growth cycle. The quantitative effects of different factors such as day/night cycle, ontogenetic stages, root temperature, photosynthetically active radiation, and soil nitrate availability on different components of the constitutive high-affinity transport systems, constitutive low-affinity transport systems, inducible low-affinity transport systems, and inducible high-affinity transport systems of nitrate were then determined to improve the model's predictions. Simulated uptake correlated well with measured values of nitrogen (N) uptake under field conditions for all N fertilization rates tested. Model outputs showed that the high-affinity transport system accounted for about 89% of total NO3- uptake (18% and 71% for constitutive high-affinity transport systems and inducible high-affinity transport systems, respectively) when no fertilizer was applied. The low-affinity transport system accounted for a minor proportion of total N uptake, and its activity was restricted to the early phase of the growth cycle. However, N fertilization in spring increased the duration of its contribution to total N uptake. Overall, data show that this mechanistic and environmentally regulated approach is a powerful means to simulate total N uptake in the field with the advantage of taking both physiologically regulated processes at the overall plant level and specific nitrate transport system characteristics into account.     

Effect of phenylphosphorodiamidate on urea hydrolysis,ammonia volatilization and rice growth in an alkali soil

Summary In order to improve nitrogen recovery by rice, the effect of a urease inhibitor phenylphosphorodiamidate (PPD) on the efficiency of fertilizer urea was studied in laboratory and greenhouse. Addition of PPD to urea (5% w/w) delayed urea hydrolysis by 3 to 4 days and reduced ammonia volatilization from 45% (without PPD) to 8.5% (with PPD). Ammonia volatilization obeyed first order kinetics. Urea hydrolysis was sufficiently strongly inhibited to match the nitrification potential of the soil. N application to rice by three different modes showed that a delayed mode (4 splits) was superior to two conventional modes (3 splits) in nitrogen recovery and fertilizer efficiency since it met nitrogen requirement of plants at reproductive stage. In 2 out of 3 modes of application, there was a 14% increase (relative) in grain yields and dry matter, and 6.8% increase in N uptake efficiency on application of PPD along with urea. The results indicate that urease inhibitors like PPD can be effectively used to block urea hydrolysis, reduce ammonia volatilization losses and improve N use efficiency by rice.     

Labeled nitrogen fertilizer research with urea in the semi-arid tropics

Summary As part of a research program to determine the fate of N fertilizers applied to dryland sorghum in the semi-arid tropics,¹⁵N balance studies were conducted with various N sources in the greenhouse. Two American soils, Houston Black clay (Udic Pellustert) and Windthorst sandy loam (Udic Paleustalf), similar in properties to the Vertisol and Alfisol in the semi-arid tropics of India, were employed. Experiments were conducted with large pots containing 20 or 60 kg of soil which was subjected to several watering regimes. The¹⁵N not accounted for in the plant and soil was presumably lost. Losses of N on calcareous Houston Black clay were greatest for broadcast urea, 16%–28%. Amendment of broadcast urea with 2% phenyl phosphorodiamidate, a urease inhibitor, reduced N losses only slightly to 15%–20%. Point placement of urea at a 6 cm soil depth reduced losses to 1%–11%. Granule size had no effect on N loss from point-placed urea. Ammonia volatilization was apparently the main N loss mechanism, since N losses from sodium nitrate were less than 7%, except when the soil surface was waterlogged. N losses on the Windthorst soil averaged 30% from urea and 11% from ammonium nitrate. Amendment of urea with urea phosphate to form a 27% N and 13% P product reduced fertilizer N losses but did not increase grain yield on Windthorst soil. N losses were also less from ammonium nitrophosphate than from urea. Band and point placement of urea 6 cm below the soil surface were equally effective in reducing N loss on Houston Black clay. The findings give credence to the recommendation of deep band placement for urea in the semi-arid tropics.     

Urea-triazone N characteristics and uses

Urea-triazone nitrogen (N) is a stable solution resulting from a controlled reaction in aqueous medium of urea, formaldehyde, and ammonia which contains at least 25% total N. This N source contains no more than 40%, nor less than 5%, of total N from unreacted urea and not less that 40% from triazone. All other N shall be derived from water-soluble dissolved reaction products of the above reactants. It is a source of slowly available N. The rate of mineralization of urea-triazone is about 66% that of urea after 8 days when incorporated in a Munjor sandy loam. Ammonia volatilization losses of N applied as urea-triazone were about 41% of those from urea on a Cecil sandy loam in the first week after application. N leaching losses through saturated Yolo loam columns of urea-triazone were about two thirds that of urea or nitrate N. This N source has proven to be a safer and more effective material for direct application on plant foliage. Tomato growth was enhanced with foliar application of urea-triazone relative to that obtained from ammonium nitrate or urea. The stability of this N source from potential losses via ammonia volatilization and nitrate leaching when soil applied is also documented by results from university trials.     

Leaf urea metabolism in potato. Urease activity profile and patterns of recovery and distribution of (15)N after foliar urea application in wild-type and urease-antisense transgenics

The influence of urease activity on N distribution and losses after foliar urea application was investigated using wild-type and transgenic potato (Solanum tuberosum cv Désirée) plants in which urease activity was down-regulated. A good correlation between urease activity and (15)N urea metabolism (NH(3) accumulation) was found. The general accumulation of ammonium in leaves treated with urea indicated that urease activity is not rate limiting, at least initially, for the assimilation of urea N by the plant. It is surprising that there was no effect of urease activity on either N losses or (15)N distribution in the plants after foliar urea application. Experiments with wild-type plants in the field using foliar-applied (15)N urea demonstrated an initial rapid export of N from urea-treated leaves to the tubers within 48 h, followed by a more gradual redistribution during the subsequent days. Only 10% to 18% of urea N applied was lost (presumably because of NH(3) volatilization) in contrast to far greater losses reported in several other studies. The pattern of urease activity in the canopy was investigated during plant development. The activity per unit protein increased up to 10-fold with leaf and plant age, suggesting a correlation with increased N recycling in senescing tissues. Whereas several reports have claimed that plant urease is inducible by urea, no evidence for urease induction could be found in potato.     

Short-term effects of urea amended with the urease inhibitor N-(n-butyl) thiophosphoric triamide on perennial ryegrass

The current study investigated the short-term physiological implications of plant nitrogen uptake of urea amended with the urease inhibitor N-(n-butyl) thiophosphoric triamide (nBTPT) under both greenhouse and field conditions. ¹⁵N labelled urea amended with 0.0, 0.01, 0.1 and 0.5% nBTPT (w/w) was surface applied at a rate equivalent to 100 kg N ha^–1 to perennial ryegrass in a greenhouse pot experiment. Root, shoot and soil fractions were destructively harvested 0.75, 1.75, 4, 7 and 10 days after fertilizer application. Urease activity was determined in each fraction together with ¹⁵N recovery and a range of chemical analyses. The effect of nBTPT amended urea on leaf tip scorch was evaluated together with the effect of the inhibitor applied on its own on plant urease activity.nBTPT-amended urea dramatically reduced shoot urease activity for the first few days after application compared to unamended urea. The higher the nBTPT concentration the longer the time required for shoot activity to return to that in the unamended treatment. At the highest inhibitor concentration of 0.5% shoot urease activity had returned to that of unamended urea by 10 days. Root urease activity was unaffected by nBTPT in the presence of urea but was affected by nBTPT in the absence of urea.Transient leaf tip scorch was observed approximately 7–15 days after nBTPT + urea application and was greatest with high concentrations of nBTPT and high urea-N application rates. New developing leaves showed no visual sign of tip necrosis.Urea hydrolysis of unamended urea was rapid with only 1.3% urea-N remaining in the soil after 1.75 days. N uptake and metabolism by ryegrass was rapid with ¹⁵N recovery from unamended urea, in the plant (shoot + root) being 33% after 1.75 days. Most of the ¹⁵N in the soil following the urea+0.5% nBTPT application was still as urea after 1.75 days, yet ¹⁵N plant recovery at this time was 25% (root+shoot). This together with other evidence, suggests that if urea hydrolysis in soil is delayed by nBTPT then urea can be taken up by ryegrass as the intact molecule, albeit at a significantly slower initial rate of uptake than NH₄ ⁺-N. Protein and water soluble carbohydrate content of the plant were not significantly affected by amending urea with nBTPT however, there was a significant effect on the composition of amino acids in the roots and shoots, suggesting a difference in metabolism.Although nBTPT-amended urea affected plant urease activity and caused some leaf-tip scorch the effects were transient and short-lived. The previously reported benefit of nBTPT in reducing NH₃ volatilization of urea would appear to far outweigh any of the observed short-term effects, as dry-matter production of ryegrass is increased.     

脲酶抑制剂和硝化抑制剂的协同作用对尿素氮转化和N2O排放的影响

根据培养试验,论述了脲酶抑制剂氢醌和硝化抑制剂双氰胺和碳化钙的不同组合在土壤正常水分和渍水的条件下对于土中尿素的水解及其释出的氨的吸附、氧化和挥发以及Ｎ２Ｏ生成的影响．文章指出,配合使用氢醌和双氰胺既能延缓土中尿素的水解并使水解后释出的氨在土中得以更多和更长时间的保持,还能减少土中硝酸盐的累积、氨挥发的损失及Ｎ２Ｏ的生成．这表明在脲酶抑制剂和硝化抑制剂间可能存在一定的协同作用．很好利用这一作用,将有益于提高尿素肥效和减少其Ｎ损失与环境污染．     

The fate of labelled15N urea and ammonium nitrate applied to a winter wheat crop

Labelled urea or ammonium nitrate was applied to winter wheat growing on a loamy soil in Northern France. Two applications of fertilizer were given: 50 kg N ha^–1 at tillering (early March) and 110 kg N ha^–1 at the beginning of stem elongation (mid-April). The kinetics of urea hydrolysis, nitrification of ammonium and the disappearance of inorganic nitrogen were followed at frequent intervals. Inorganic nitrogen soon disappeared, mainly immobilized by soil microflora and absorbed by the crop. Net immobilization of fertilizer N occured at a very similar rate for urea and ammonium nitrate. Maximum immobilization (16 kg N ha¹) was found at harvest for the first dressing and at anthesis for the second dressing (23 kg N ha¹). During the nitrification period, the labelled ammonium pool was immobilized two to three times faster than the labelled nitrate pool. No significant net¹⁵N remineralization was found during the growth cycle.The actual denitrification and volatilization losses were probably more important than indicated from calculations made by extrapolation of fluxes measured over short intervals. However microbial immobilization was the most important of the processes which compete with plant uptake for nitrogen.     

渍水对油菜苗期生长及生理特性的影响

通过盆栽试验研究了模拟渍水条件对苗期油菜（Brassica napus L.）中双11号生长及生理特性的影响。结果表明,渍水超过3 d就会显著抑制苗期油菜的生长,这与渍水降低油菜叶片叶绿体和光合色素含量、降低氮素吸收利用和引起过氧化胁迫有关。渍水后苗期油菜的生理恢复,无论是叶绿体和光合色素含量,还是硝酸还原酶及抗氧化酶活性的恢复均表现出一定的滞后性,而且渍水时间越长滞后期越长。此外,在苗期油菜叶片光合色素中,渍水对类胡萝卜素的影响要大于对叶绿素的影响。     

深松与包膜尿素对玉米田土壤氮素转化及利用的影响

耕作方式和氮肥施用是影响土壤中氮肥转化、利用效率和作物产量的重要因素。通过夏玉米田的2a(2011—2012)定位试验,研究了两种耕作方式(深松、旋耕)配合不同尿素类型(包膜尿素、普通尿素)的施用对玉米田土壤硝态氮和铵态氮含量、脲酶活性、硝化细菌和反硝化细菌数量、玉米产量以及氮肥农学效率的影响。研究结果表明:相同耕作方式下,包膜尿素处理土壤中脲酶活性较稳定,且增加了旱田土壤亚硝酸细菌数量而降低了反硝化细菌数量,有利于土壤硝态氮含量的提高,尤其是作物生长的中后期;包膜尿素处理的产量比普通尿素提高7.25%—10.82%,同时提高氮肥农学效率。深松处理增加了土壤中的反硝化细菌数量,配合施用包膜尿素进一步提高了土壤脲酶活性,增加了亚硝酸细菌数量;旋耕与包膜尿素配合施用在一段时期内能显著增加土壤硝态氮含量,减少反硝化细菌数量。深松配合包膜尿素处理能够显著的增加玉米产量,2a分别比旋耕配合包膜尿素增加1.41%和10.62%。因此,深松措施配合施用包膜尿素能够增强土壤脲酶活性,增加亚硝酸细菌数量,提高氮素转化速率,增加作物产量和氮肥农学效率,其稳产效果在干旱年份尤为显著。     

Nitrogen efficiency and leaf nitrogen remobilisation of oilseed rape lines and hybrids

Improved nitrogen (N) efficiency of oilseed rape is crucial for reducing environmental N surpluses. In this study, a 2‐year field experiment as well as a hydroponic experiment were performed with four hybrids and their corresponding lines. Seed yield and N efficiency parameters of oilseed rape cultivars grown at low N (0 kg N ha^?1) and high N (150 kg N ha^?1) supply were investigated in the field experiments. Hybrids showed higher seed yield than lines, especially at low N supply, because of a superior N uptake. Moreover, hybrids showed higher N harvest index (NHI) across N rates, which also contributed to higher seed yields. Results from the hydroponic experiment showed significant genotypic variation in leaf N remobilisation efficiency (NRE), but no hybrid‐versus‐line difference was found. Cultivars differed significantly in specific N content in senescent leaves, and leaf NRE was negatively correlated with specific N content in senescent leaves. When linking the hydroponic results with the field results, no relationship was found between leaf NRE and NHI. In conclusion, hybrids were superior to their corresponding lines in N efficiency because of higher N uptake and NHI. The higher NHI was, however, not related to genotypic variation in leaf NRE.     

Inhibition of endogenous urease activity by NBPT application reveals differential N metabolism responses to ammonium or nitrate nutrition in pea plants: a physiological study

Background and aims

Urea is the predominant form of N applied as fertilizer to crops, but it is also a significant N metabolite of plants themselves. As such, an understanding of urea metabolism in plants may contribute significantly to subsequent N fertilizer management. It currently appears that arginase is the only plant enzyme that can generate urea in vivo. The aim of this work was, therefore, to gain a more in-depth understanding of the significance of the inhibition of endogenous urease activity and its role in N metabolism depending on the N source supplied.

Methods

Pea (Pisum sativum cv. Snap-pea) plants were grown with either ammonium or nitrate as the sole N source in the presence or absence of the urease inhibitor NBPT.

Results

When supplied, NBPT is absorbed by plants and translocated from the roots to the leaves, where it reduces endogenous urease activity. Different N metabolic responses in terms of N-assimilatory enzymes and N-containing compounds indicate a different degree of arginine catabolism activation in ammonium- and nitrate-fed plants.

Conclusions

The arginine catabolism is more highly activated in ammonium-fed plants than in nitrate-fed plants, probably due to the higher turnover of substrates by enzymes playing a key role in N recycling and remobilization during catabolism and in early flowering and senescence processes, usually observed under ammonium nutrition.     

The fate of spring applied fertilizer N during the autumn-winter period: comparison between winter-fallow and green manure cropped soil

A field experiment was carried out at a pilot plot that was cropped with oilseed rape, and then left partly fallow and partly cropped with a green manure (mustard) during the autumn after harvest of the oilseed rape. The rape residues were incorporated in the soil. Methods used to quantify the N fluxes from harvest until sowing of the next crop were (1) ¹⁵N balance method, (2) total mineral N analysis and (3) NO emission measurements. Losses of spring applied fertilizer N were negligible in cropped plots and minimal in fallow plots during the following autumn-winter period. Most of the plant-N residues was retained by the organic N pool of the upper 30-cm soil layer. The green manure contributed slightly to soil available N at sowing of the next crop. However, the incorporation of plant material resulted in a nitrate flux that was at risk of leaching on the fallow plots, and on the green manure plots after incorporation of the green manure. This nitrate was largely derived from soil organic N, not from unused fertilizer applied in spring or from immobilized fertilizer. The NO emissions from the green manure plots were significantly higher than emissions from the fallow plots. The plants had a stimulating effect on the NO emission. A relationship between the NO emission and the soil nitrate concentration could not be established. No emissions were measured after green manure incorporation due to the low temperatures at the pilot plot. However, a greenhouse experiment showed an increased emission after incorporation. The NO emissions seemed to be related with the soil ammonium concentration.     

Integrated Control of Nitrate Uptake by Crop Growth Rate and Soil Nitrate Availability under Field Conditions

There is still disagreement about whether crop growth rate orsoil nitrate concentration control nitrogen absorption by cropsunder field conditions. The influence of these factors on thecontrol of N uptake rate was examined in the absence of waterstress, using data on dry matter production, above-ground nitrogenaccumulation and soil nitrate concentration from several N-fertilizerexperiments on winter wheat, winter oilseed rape and maize.The results confirmed that crops can accumulate nitrogen farin excess of the ‘critical dilution curve’, whichdefines the minimum amount of nitrogen needed for maximal growthrate: the N concentration in plants could exceed the criticalN concentration by 70 to 80% for the three species studied.The nitrate uptake rate index (NUI) was calculated as the ratioof actual and critical N uptake rates, at intervals of 1 week.NUI varied with nitrate concentration in the 0–30 cm soillayer according to a Michaelis–Menten equation (with oneor two components). This response was compared with the kineticsof saturation of the nitrate uptake systems: the high affinitytransport system (HATS) and the low affinity transport system(LATS). As a result, it is proposed that there is a criticalN dilution curve delimiting two domains of N use by plants.This is linked to the two nitrate transport systems, with HATSworking at low nitrate concentrations, below the critical dilutioncurve, and LATS at high nitrate concentrations, above the curve.NUI provides another method for calculating the actual nitrateuptake rate, which depends on the maximal crop growth rate (withoutN deficiency) and on the external nitrate concentration. Copyright2000 Annals of Botany Company Nitrate, uptake rate, growth rate, wheat, maize, oilseed rape, soil N availability     

硝酸还原酶抑制剂钨酸钠对油菜硝态氮积累的影响

采用溶液培养方法,选取硝酸盐积累差异明显的两个油菜品种（低硝态氮积累品种‘红油3号’和高硝态氮积累品种‘中双6号’,研究苗期根系硝酸还原酶（NR）活性被抑制以后两个油菜品种叶片、叶柄和根系中NR活性和硝态氮含量的变化。结果表明：1.0mmol.L-1的NR活性抑制剂Na2WO4对两个油菜品种的根系NR活性抑制效果最佳;根系NR活性被抑制以后,两个油菜品种的根系NR活性、硝态氮吸收速率均显著下降,而硝态氮含量却显著上升;且Na2WO4对‘中双6号’硝态氮吸收的抑制程度强于其对‘红油3号’的抑制。叶片和叶柄的NR活性变化不显著,但叶柄硝态氮含量显著下降,叶片硝态氮含量稳定,且这一趋势在低积累品种‘红油3号’中表现得更为明显。