TY - JOUR
AU - Ourry, Alain
AB - Abstract N-fertilizer use efficiencies are affected by their chemical composition and suffer from potential N-losses by volatilization. In a field lysimeter experiment, 15N-labelled fertilizers were used to follow N uptake by Brassica napus L. and assess N-losses by volatilization. Use of urea with NBPT (urease inhibitor) showed the best efficiency with the lowest N losses (8% of N applied compared with 25% with urea alone). Plants receiving ammonium sulphate, had similar yield achieved through a better N mobilization from vegetative tissues to the seeds, despite a lower N uptake resulting from a higher volatilization (43% of applied N). Amounts of 15N in the plant were also higher when plants were fertilized with ammonium nitrate but N-losses reached 23% of applied N. In parallel, hydroponic experiments showed a deleterious effect of ammonium and urea on the growth of oilseed rape. This was alleviated by the nitrate supply, which was preferentially taken up. B. napus was also characterized by a very low potential for urea uptake. BnDUR3 and BnAMT1, encoding urea and ammonium transporters, were up-regulated by urea, suggesting that urea-grown plants suffered from nitrogen deficiency. The results also suggested a role for nitrate as a signal for the expression of BnDUR3, in addition to its role as a major nutrient. Overall, the results of the hydroponic study showed that urea itself does not contribute significantly to the N nutrition of oilseed rape. Moreover, it may contribute indirectly since a better use efficiency for urea fertilizer, which was further increased by the application of a urease inhibitor, was observed in the lysimeter study. Ammonium, BnAMT1.1, BnDUR3, BnNRT1.1, BnNRT2.1, Brassica napus, 15N labelling, N losses, nitrate, uptake, urea, urease inhibitor Introduction Nitrogen (N) is the most important plant nutrient for crop production and it is generally applied to the soil as a fertilizer. Three main forms of mineral N fertilizers are usually available as ammonium (NH4+), as nitrate (NO3–) and as urea [CO(NH2)2] or in combined forms, and their effectiveness is influenced by the ion exchange principles. Because of its positive charge, NH4+ is adsorbed by negatively charged soil colloids and are thus protected from leaching, whereas the negatively charged NO3– is subject to leaching (Hofman and Van Cleemput, 2004). However, depending on many factors, such as soil characteristics, climatic factors, crop type, and fertilization management, the N-use efficiency of plants is generally low (Malhi and Nyborg, 1991; Malhi et al., 2001). Urea is the main N fertilizer used in agriculture throughout the world, accounting for about 50% of the total world N fertilizer consumption (http://faostat.fao.org). Its consumption has increased substantially because of its low manufacturing cost and its high nitrogen content (46%). However, after its hydrolysis by urease, a common enzyme in soil, the efficiency of urea can decrease significantly due to losses of N as ammonia gas (NH3), which may account for as much as 50% of the N applied (Terman, 1979). Thereafter, NH3 lost to the atmosphere will be deposited on land or water causing eutrophication and acidification of natural ecosystems (Sommer and Hutchings, 2001). The extent of ammonia volatilization can be affected by pH, temperature, organic mater, the availability of water, and the form in which urea is applied (Vlek and Carter, 1983). Moreover, the urea hydrolysis reaction leads to an increase in soil pH and to the accumulation of ammonium and nitrite, especially in alkaline sandy soils and with urea surface application (Trenkel, 2010). Nitrite accumulation could be toxic for germinating seeds and seedlings, and it could also favour gaseous N losses by denitrification (Bremner and Chai, 1989). To reduce ammonia volatilization and the adverse effect of urea hydrolysis, many compounds have been tested for their ability to inhibit urease activity (Trenkel, 2010). The use of urease inhibitors, which delay the rate at which urea is hydrolysed and converted to ammonium, may reduce the problems associated with the use of urea-based fertilizer (Gill et al., 1997). By slowing down urea hydrolysis, atmospheric volatilization of ammonia, as well as further losses from nitrate leaching, are either reduced or avoided. It also allows more time for plant N uptake. Amongst the inhibitors tested, N-(n-butyl)thiophosphoric triamide (NBPT, sold as Agrotain®, Agrotain International) was found to be one of the most effective (Sanz-Cobena et al., 2008; Trenkel, 2010). It has consistently demonstrated its effectiveness at low concentrations, and inhibits the activity of the urease enzyme in a wide variety of soils (Carmona et al., 1990; Antisari et al., 1996; Gill et al., 1997; Trenkel, 2010). Its effect on urease activity is associated with the activity of its derivative, the oxygen analogue, N-(n-butyl) phosphoric triamide (Phongpan et al., 1995). Studies on soil incubation show that the effect of NBPT on lowering ammonia volatilization was greatest in soils with a high pH and a low buffering capacity (Watson et al., 1994). In addition, compared with urea without inhibitor, there are decreases in nitrogen losses and reduced toxicity from ammonia resulting in larger yields and better crop quality when NBPT is applied to the soil together with urea fertilizer (Trenkel, 2010). The use of a urease inhibitor implies that a significant soil concentration of stable urea and ammonium, as well as new mixed N forms such us NH4+–NO3– (AN) and NO3– urea (NU) are available for plants besides NO3–, which is the main N form present in cultivated soil (Marschner, 1995). All plants are able to take up either nitrate, ammonium or urea as a nitrogen source, and possess dedicated transmembrane transport systems in root cells for each form of nitrogen (Forde, 2000; Liu et al., 2003). Recently, AtDUR3 was demonstrated to serve as a major high affinity transporter for urea uptake by N-deficient roots of Arabidopsis (Kojima et al., 2007), suggesting that urea transport activity of AtDUR3 is of physiological significance in higher plants. Ammonium uptake is mediated by transport systems that have been isolated and partially characterized in several plant species, such as Arabidopsis thaliana (AtAMT1.1, AtAMT1.2, AtAMT1.3, and AtAMT2; Ninnemann et al., 1994; Gazzarrini et al., 1999; Sohlenkamp et al., 2000; Kaiser et al., 2002) and Brassica napus (BnAMT1.2; Pearson et al., 2002). Two gene families, namely NRT1 and NRT2, have been identified in nitrate acquisition by plants (Forde, 2000). In Arabidopsis thaliana, AtNRT1.1 and AtNRT1.2 were characterized as low-affinity nitrate transporters with constitutive expression and were considered to belong to the NO3– LATS (Low Affinity Transport System; Crawford and Glass, 1998), while the NRT2.1 gene was shown to encode a major component of the NO3– HATS (High Affinity Transport System; Cerezo et al., 2001). Recently, it has been shown that NRT1.1 is involved in the signalling pathway responsible for the stimulation of lateral root growth and that it facilitates the uptake of auxin (Krouk et al., 2010). It has been established that the efficiency of urea as a source of N seems to be lower than that of NO3– and NA (Tan et al., 2000; Houdusse et al., 2005; Mérigout et al., 2008). NH4+ nutrition usually has deleterious effects on plant growth and can result in toxicity symptoms in many plants (Britto and Kronzucker, 2002). Therefore, NH4+ must be rapidly assimilated. It is widely accepted that the glutamine synthetase (GS)-glutamate synthase (GOGAT) cycle is the main pathway of NH4+ assimilation (Lea and Miflin, 1974). However, glutamate dehydrogenase (GDH) may play a complementary role to GS/GOGAT in the reassimilation of ammonium during stress conditions or during specific stages of development (Yamaya et al., 1986; Rhodes et al., 1989; Terce-Laforgue et al., 2004). Experiments conducted in controlled conditions have shown that urea nutrition can also cause negative effects on wheat development, but less intensely than those associated with NH4+ nutrition (Houdusse et al., 2005; Garnica et al., 2009). However, several authors have demonstrated that the negative effects associated with ammonium and urea nutrition are corrected by the supply of nitrate in the nutrient solution (Britto and Kronzucker, 2002; Cruz et al., 2003; Houdusse et al., 2005; Garnica et al., 2009). Physiological and molecular processes underlying this beneficial effect are only partially understood. The influences of both the urease inhibitor and the type of fertilizer on yield and N uptake described above indicate that these two parameters could influence N use efficiency as well as the N management of agricultural systems, and are potentially of importance from the perspectives of pollution, cost, and plant productivity. In this respect, oilseed rape (Brassica napus L.), which is the dominant oilseed crop in northern Europe, requires high amounts of nitrogen (N) but is characterized by low real harvested N-fertilizer use efficiency. It is considered a nitrophilic crop, and receives a large amount of N fertilizer. Also, through a European Directive (2001/81/EC) and the Gothenburg Protocol, France has committed to a reduction in ammonia emissions. Therefore, there is a strong need to review all the relevant information on the N metabolism of oilseed rape to improve the efficiency of its production and reduce its environmental impact. The aims of this work were firstly to investigate, on undisturbed soil lysimeters, the effect of different types of nitrogen fertilizers on crop yield of spring oilseed rape (Brassica napus L.) and their impacts on the environment. Assessment of NH3 volatilization was determined by 15N balance. This would allow a clearer assessment of the significance of urea fertilization combined or not combined with the urease inhibitor, NBPT, compared with a commonly used fertilizer such as ammonium nitrate. In addition to field studies, the effects of mixed feeding (AN and NU) on the main nitrate (NRT1.1 and NRT2.1), ammonium (AMT1.1), and urea (DUR3) transporters compared with nitrate, urea, and ammonium as the sole nitrogen source was studied at the physiological and molecular levels by the use of 15N labelling and qPCR expression analysis of BnNRTt1.1, BnNRT2.1, BnDUR3, BnAMT1.1, and BnGDH2. Moreover, experiments on the possible role of nitrate as a signal responsible for growth were undertaken. One of the aims of the laboratory experiments was to identify the mechanisms responsible for the patterns of N uptake observed in the field. Materials and methods Site and lysimeter description The lysimeter system is located in Lieury l’Oudon, in the North of France (00°00'34.3'' W, 48°59'24.2''N). It is composed of 15 lysimeter boxes, each composed of polyester resin with an area of 2 m2 and a depth of 110cm containing a 10cm thick base of fine stone for drainage. Each lysimeter box is connected to a bucket collecting water percolation through a PVC tube. All buckets are located in a concrete tunnel below the boxes. Soil and crop culture The soil in the lysimeter boxes is a Calcaric Cambisol (World Reference Base for soil resources, 2006), with a fertile surface layer in terms of organic matter (6.3% in 0–20cm), a relatively basic pH, and a high content of calcium carbonate increasing with depth (Table 1). A spring oilseed rape crop (Brassica napus L. ‘Seven’) was sown on 9 March 2011 at a density of 100–120 seeds m–2, which was then controlled throughout the crop cycle (from plant emergence to harvest), to obtain a plant density of between 77–81 plants m–2. The monthly water input, including rainfall and irrigation, and the monthly average temperature are shown in Fig. 1. Water stress was not observed during the experimental period due to regular irrigation during March, April, May, and June, while important rainfalls during July and August were observed. Experimental treatment, labelling, and harvest A total of four 15N-labelled fertilizers (15N excess=10%), each with three replicates, were allocated to the lysimeters in a randomized design: 15NH415NO3 (Ammonium Nitrate, AN), (15NH4)2SO4 (Ammonium Sulphate, AS), CO(15NH2)2 (Urea, U), CO(15NH2)2+NBPT (Urea+N-(n-butyl) thiophosphoric triamide, U+NBPT), and a control (treatment without fertilizer). Fertilizers were applied 1 month after sowing (in the middle of April) at the GS 1.4 stage of development, and at a rate of 100kg N ha–1. To facilitate the uniformity of application, fertilizer was dissolved in deionized water and spread with a watering can. After application, leaves were rinsed with deionized water to prevent burns. At harvest, on 10 August, plants were collected, weighed (fresh weight, FW), and subdivided according to their component parts (seeds, senescent leaves, stems, and tap roots) and dried (dry weight, DW). Weeds and senescent leaves were also collected during the developmental cycle, weighed (FW), and dried (DW). For soil analysis, five cores per lysimeter were obtained down to 0.85 m. After drying at 60 °C, samples were weighed and ground to a fine powder with inox beads of 0.4mm diameter in an oscillating grinder (mixer mill MM301; Retsch) before isotope analysis. Plant material for laboratory experiments Seeds of Brassica napus L. were surface-sterilized by exposure to 80% ethanol for 30 s followed by 20% sodium hypochlorite for 10min. After 10 washes with demineralized water, seeds were germinated on perlite over deionized water for 2 d in darkness. Next, plants were grown in the greenhouse. Just after the first leaf emergence, seedlings were transferred for 1 week to a plastic tank (20 l) and supplied with a continuously aerated nutrient solution containing: CaCl2 1.25mM, KCl 0.25mM, KH2PO4 0.25mM, MgSO4 0.5mM, EDTA-2NaFe 0.2mM, H3BO3 14 µM, MnSO4 5 µM, ZnSO4 3 µM, CuSO4 0.7 µM, (NH4)6Mo7024 0.7 µM, CoCl2 0.1 µM, and NiCl2 0.04 µM. Next, seedlings were divided into seven sets and placed in a plastic tank (10 l, 32 seedlings) containing the nutrient solution described above and supplied with 2mM N-CO(15NH2)2 [U; atom% 15N, 4.11%), N-(15NH4)2SO4 (AS; atom% 15N, 2.29%), N-K15NO3 (Nitrate; atom% 15N, 2.25%), K15NO3-CO(NH2)2 (NU; atom% 15N, 2.32%), KNO3-CO(15NH2)2 (NU; atom% 15N, 5.17%), (NH4)2SO4-K15NO3 (AN; atom% 15N, 2.49%) and (15NH4)2SO4-KNO3 (AN; atom% 15N, 2.96%) to measure cumulative nitrogen uptake. Two other sets of seedlings were treated with 2mM N-CO(15NH2)2 (U; atom% 15N, 4.11%) or 2mM N-(15NH4)2SO4 (AS; atom% 15N, 2.29%) and received 150nM of nitrate (KNO3) every 6 h as a pulse until the end of the experiment. The natural light from the greenhouse was supplemented with high pressure sodium lamps (Philips, Master Green Power T400W) supplying an average photosynthetically active radiation of 280 µmol photons m–2 s–1 at canopy height for 16h d–1. The thermoperiod was 20 °C (day) and 17 °C (night), and the nutrient solution was renewed every 2 d. After different durations of treatment (0, 24, 72h, and 15 d), plants were harvested and separated into shoot and root samples. They were then weighed (FW), frozen in liquid nitrogen, and stored at –80 °C for further analysis. An aliquot of each tissue was weighed and dried (60 °C) in a drying oven for dry weight (DW) determination and ground to a fine powder with 0.4mm diameter inox beads in an oscillating grinder (mixer mill MM301; Retsch) before isotope analysis. Total N, 15N analysis, and 15N balance An aliquot of each collected compartment sample (from soil and plants) was placed in a tin capsule for 15N isotopic analysis. The total N amount and 15N excess in plant or soil samples were determined with a continuous flow isotope mass spectrometer (Isoprime, GV Instruments, Manchester, UK) linked to a C/N/S analyser (EA3000, Euro Vector, Milan, Italy). Total N (Ntot) content in the different compartments ‘i’ was calculated as: Ntot = (%Ni×DWi)/100. The natural 15N abundance of atmospheric nitrogen (0.36636%) was used as a reference for 15N analysis of plants grown in hydroponics. Because lysimeters have been used for 15N labelling experiments for 10 years now, the natural abundance of control plants (available from non-fertilized treatments, 0.3923%) was used as a reference for 15N analysis of plants grown under the lysimeter conditions. The natural 15N abundance in unfertilized lysimeter soil (0.3816%) was used as a reference for soil analysis. Nitrogen derived from current N uptake (Nupti) in ‘i’ compartment was calculated as: Nupti = (Ntoti×Ei)/Es where Ei (%) is the atom 15N excess in the different compartments, and Es is the nutrient solution or fertilizer atom 15N excess. Fertilizer Use Efficiency (FUE) was calculated as: 15Nuptake/15Napplied × 100, while nitrogen losses by volatilization were estimated using the 15N balance and calculated by the following formula: 15Nvolatilized = 15Nfertilizer – (15Nplant + 15Nsoil + 15Npercolated). RNA isolation and quantitative RT-PCR analysis Total RNA was extracted from 200–400mg of fresh root and shoot matter corresponding to three sets of seedlings for each treatment. Fresh root and shoot samples were ground in liquid nitrogen with a mortar. The resulting powder was suspended in 750 µl of extraction buffer (0.1M TRIS, 0.1M LiCl, 0.01M EDTA, and 1% SDS w/v, pH 8) and 750 µl of hot phenol (80 °C, pH 4). This mixture was vortexed for 30 s. After the addition of 750 µl of chloroform/isoamylalcohol (24:1 v/v), the homogenate was centrifuged (15 000 g, 5min, 4 °C). The supernatant was transferred into 4M LiCl solution (w/v) and incubated overnight at 4 °C. After centrifugation (15 000 g, 30min, 4 °C), the pellet was suspended in 250 µl of sterile water. Fifty µl of 3M sodium acetate (pH 5.6) and 1ml of 96% ethanol were added to precipitate the total RNA for 1h at –80 °C. After centrifugation (15 000 g, 20min, 4 °C), the pellet was washed with 1ml of 70% ethanol, then centrifuged at 15 000 g for 5min at 4 °C. The resulting pellet was dried for 5min at room temperature and re-suspended in sterile water containing 0.1% SDS and 20mM EDTA. Quantification of total RNA was performed with a spectrophotometer at 260nm (BioPhotometer Eppendorf, France) before RT-PCR analysis. For RT, 1 µg of total RNA was converted to cDNA with an iScript cDNA synthesis kit using the manufacturer’s protocol (Bio-Rad). The genes and specific primers selected for the analysis were as follows: EF1 [Forward (F), 5'-tttcgagggtgacaacatga; Reverse (R), 5'-ccgttccaataccaccaatc); and 18S (F, 5'-cggataaccgtagtaattctag; R, 5'-gtactcattccaattaccagac); as housekeeping genes. BnNrt2.1 (F, 5'-tggtggaataggcggctcgagttg; R, 5'-gtatacgttttgggtcattgccat); BnNrt1.1 (F, 5'-atggtaaccgaagtgccttg; R, 5'-tgattccagctgttgaagc); BnDUR3 (F, 5'-gacgacgagggaaatcaaag; R, 5'-atgacaacaatgaggagagtgaa); and BnGDH2 (F, 5'-ctcgtgacttgagcttgagc; R, 5'-caggggaatgaccatgaaac); as target genes. The subsequent PCR reactions were performed with 4 µl of cDNA diluted ×200, 500nM of the primers, and 1× SYBR Green PCR Master Mix (Bio-Rad) in a total volume of 15 µl. The specificity of PCR amplification was examined by monitoring the melting curves after quantitative PCR reactions using the Chromo4 system (Bio-Rad, France) and by sequencing the quantitative PCR product to confirm that the correct amplicons were produced from each pair of primers (Biofidal, France). For each sample, the subsequent Q-PCR reactions were performed in triplicate and the relative expression of the target gene in each sample was compared with the control sample (corresponding to control plants at T=0) and was determined with the delta-delta Ct method using the following equation: 2–[ΔCt sample–ΔCt control], with ΔCt=Cttarget gene–Cthousekeeping gene (for calculations, we considered the geometric mean between Ct of the two housekeeping genes), where Ct refers to the threshold cycle determined for each gene in the exponential phase of PCR amplification. Using this analysis method, the relative expression of the target gene in the control sample was equal to one (20), by definition (Livak and Schmittgen, 2001). Data and statistical analysis All experiments were performed with three replicates. For the laboratory experiments, each replicate contained 32 seedlings. The resulting variation in the measurements was expressed as the mean ±S.D. for n=3. A statistical analysis was performed using the Student test (P=0.05). For qPCR analysis, the normality of the data was studied with the Ryan–Joiner test at 95%. Analysis of variance (ANOVA) and the Tukey test to compare the means were performed using MINITAB13 for Windows (Minitab Inc, State College, PA, USA). When the normality law of the data was not respected, the non-parametric test of Kruskal–Wallis was carried out. Statistical significance was postulated at P <0.05. Results Fertilizer net uptake and N partitioning under lysimeter conditions Environmental conditions Monthly air temperature at the experimental site varied from 7.6 °C in March to 17.6 °C in August (Fig. 1). Cumulative water input, including rainfall and irrigation, was 24mm at the beginning of the experiment, and reached 71.1, 58.3, and 89.3mm during May, June, and July, respectively. The highest rate of rainfall was recorded at the end of the experiment (August) with 116.4mm (Fig. 1). During the spring and summer periods, the irrigation and rainfall inputs were mostly lost to the atmosphere as evapotranspiration, and no leaching occurred during this period. Fig. 1. View largeDownload slide Monthly water inputs (rainfall and irrigation) and monthly average air temperature recorded at the lysimeter site. Fig. 1. View largeDownload slide Monthly water inputs (rainfall and irrigation) and monthly average air temperature recorded at the lysimeter site. Yield and fertilizer use efficiency (FUE) A lysimeter device was used to investigate the effect of different types of nitrogen fertilizer and to assess the significance of urea fertilization combined or not combined with the urease inhibitor on yield of oilseed rape (Brassica napus L.), N uptake, and their impact on the environment. Results of crop yield, expressed in tonnes per hectare, revealed highly significant differences between control (without N fertilizer) and other treatments [Urea, U; Urea+N-(n-butyl) thiophosphoric triamide, U+NBPT; Ammonium Sulphate, AS, and Ammonium Nitrate, AN; Fig. 2A]. Compared with the control, fertilized plants were characterized by a 35% increase in yield (from 1.72±0.04 to 2.31 tonnes ha–1 on average; Fig. 2A). Results also show that the type of nitrogen fertilizer applied to plants has no effect on yield since the final crop yield did not change (2.31 tonnes ha–1 on average), whatever the fertilizer treatments. However, fertilizer use efficiency (FUE), which measures the efficiency of nitrogen uptake by plants from fertilizer, varied depending on the fertilizer provided (Fig. 2B). In fact, AS treatment has the lowest FUE (29%), while U+NBPT treatment showed the highest FUE (46%). U and AN treatments had a FUE of 37% and 41%, respectively (Fig. 2B). Furthermore, compared with the urea fertilizer, an increase of 24% in FUE was observed when the urease inhibitor was combined with urea (U+NBPT treatment). Fig. 2. View largeDownload slide Agronomic parameters. (A) Yield of oilseed rape and (B) Fertilizer Use Efficiency (FUE). Fertilizers were applied as Ammonium Nitrate (AN), Ammonium Sulphate (AS), Urea (U), Urea+N-(n-butyl) thiophosphoric triamide (U+NBPT), and a control (treatment without fertilizer). Data represent mean ±standard deviation (n=3). Asterisks represent significant differences from the control at *P <0.05 or **P <0.01. Fig. 2. View largeDownload slide Agronomic parameters. (A) Yield of oilseed rape and (B) Fertilizer Use Efficiency (FUE). Fertilizers were applied as Ammonium Nitrate (AN), Ammonium Sulphate (AS), Urea (U), Urea+N-(n-butyl) thiophosphoric triamide (U+NBPT), and a control (treatment without fertilizer). Data represent mean ±standard deviation (n=3). Asterisks represent significant differences from the control at *P <0.05 or **P <0.01. Partitioning of N following uptake By labelling fertilizers with 15N it was possible to quantify N distribution in each organ compartment (Fig. 3A), accumulated N uptake, and allocation to each plant part (Fig. 3B). The amount of residual 15N in soil was highest in the U+NBPT treatment and lowest in the AS treatment, with 45.44±4.77 and 26.97±6.05kg 15N ha–1, respectively (Fig. 3A). Compared with plants receiving urea (U, 36.43±2.87kg 15N ha–1) as fertilizer, those grown in the presence of a urease inhibitor (U+NBPT treatment) were characterized by a larger nitrogen content in the plant compartment (+23%, 44.98±3.38kg 15N ha–1), while those receiving AS have the lowest levels (28.73±0.51kg 15N ha–1, Fig. 3A). The amounts of 15N in the plant organ compartments were also increased compared with AS when plants were fertilized with ammonium nitrate (AN), with a nitrogen content of 40.86±4.04kg 15N ha–1. Fig. 3. View largeDownload slide N-fertilizer uptake and partitioning estimated by 15N-labelling. (A) Partitioning of 15N among the different compartments (soil, plant and volatilized form). (B) Distribution of 15N into the plants and their components (seeds, senescent leaves, stems, and tap roots). Fertilizers were applied as Ammonium Nitrate (AN), Ammonium Sulphate (AS), Urea (U), Urea+N-(n-butyl) thiophosphoric triamide (U+NBPT), and a control (treatment without fertilizer). Data represent mean ±standard deviation (n=3). Asterisks represent significant differences from the control at *P <0.05 or **P <0.01. Fig. 3. View largeDownload slide N-fertilizer uptake and partitioning estimated by 15N-labelling. (A) Partitioning of 15N among the different compartments (soil, plant and volatilized form). (B) Distribution of 15N into the plants and their components (seeds, senescent leaves, stems, and tap roots). Fertilizers were applied as Ammonium Nitrate (AN), Ammonium Sulphate (AS), Urea (U), Urea+N-(n-butyl) thiophosphoric triamide (U+NBPT), and a control (treatment without fertilizer). Data represent mean ±standard deviation (n=3). Asterisks represent significant differences from the control at *P <0.05 or **P <0.01. Nitrogen losses by volatilization were assessed using the 15N balance in the absence of any leaching. The results showed that, compared with U, AS, and AN treatments, the application of the urease inhibitor, NBPT, with the urea fertilizer substantially reduced the losses of nitrogen by volatilization (Fig. 3B). In fact, with NBPT, losses were reduced by 68% when compared with the U treatment (from 25.60±6.94 to 8.07±1.89kg 15N ha–1), and by 81% and 65% when compared with AS (43.33±6.11kg 15N ha–1) and AN (23.58±3.31kg 15N ha–1, Fig. 3A) treatments. N that remained in the soil can be interpreted as the results of the volatilization process and plant uptake. No significant differences were observed in 15N-soil content between the U and AN treatments (36.62±5.33 and 34.06±5.64kg 15N ha–1, respectively), while this parameter was the lowest in AS fertilized plants (Fig. 3B). The quantification of labelled N into each organ revealed that the distribution of nitrogen in the plant varies with the type of fertilizer. Overall, most of the 15N was allocated to the seeds, while the tap root, stems, and senescent leaves had the lowest 15N content (Fig. 3B). Regarding taproots, and compared with U (2.52±0.28kg 15N ha–1), U+NBPT (2.86±0.28kg 15N ha–1), and AN (2.65±0.28kg 15N ha–1) treatments, AS-treated plants were characterized by the lowest 15N content (1.87±0.28kg 15N ha–1; Fig. 3B). The results also showed that 15N content decreased significantly in stems (–30%) and senescent leaves (–36%) of plants amended with ammonium sulphate (AS), compared with those that received urea as fertilizer. Furthermore, compared with the same treatment (U), stem and senescent leaf-15N content increased in U+NBPT and AN-treated plants. However, this increase was higher with the U+NBPT treatment (+29% for stem and senescent leaves, from 2.56±0.13 to 3.32±0.11kg 15N ha–1 and from 3.58±0.48 to 4.63±0.24kg 15N ha–1, respectively). Similar trends were found in seeds, with a significant increase in 15N content in the U+NBPT-treated plants (+22%, from 28.05±2.09 to 34.42±2.91kg 15N ha–-1), and a general decrease in the AS (–18%) and AN (–4%)-treated plants (Fig. 3B). Taken together, these results show that with a lower N availability resulting from higher volatilization (AS treatment), a similar seed yield was achieved by a better mobilization of N from senescing leaves, stems and taproots to the seeds. This increased mobilization of nitrogen under ammonium nutrition can be also substantiated by the fact that only 7.7% of 15N taken-up remained in leaves versus more than 10.4% in U+NBPT-treated plants. In addition, ammonium supply may have resulted in higher N mineralization, making more unlabelled N available for root uptake. This hypothesis cannot be ruled out. Nitrate, ammonium, and urea net uptake under controlled conditions Dry matter and N content In order to study the mechanisms of nitrate’s beneficial effect, rapeseed seedlings were grown with diluted Hoagland solution without nitrogen for 1 week, and then supplied (T=0) with different nitrogen compounds in sole (Urea, U; Ammonium Sulphate, AS, and Nitrate) or mixed forms: Ammonium-Nitrate (AN) and Nitrate-Urea (NU) for 24h, 72h and 15 d. Urea analysis of the nutrient solution was regularly performed to check that no significant hydrolysis occurred. Plant growth (i.e. dry matter production) was not significantly affected during the first 24h and 72h of treatment (data not shown). After 15 d of treatment, the analysis of the accumulated dry biomass revealed highly significant differences (Fig. 4A). Compared with nitrate-fed plants, plants treated with ammonium (AS) were characterized by a 50% decrease in shoot and root dry weights after 2 weeks (from 1.37±0.12 to 0.68±0.03g DW.plant–1). This effect was more pronounced when plants were treated with urea (U) and symptoms of N starvation appeared. In fact, the growth was reduced by 75% when urea was supplied as a sole N source (from 1.37±0.12 to 0.35±0.01g DW plant–1). The results also showed that the addition of nitrate to urea (NU) or ammonium (AN) fed plants significantly reduced this negative effect compared with the provision of either N source alone, since plant dry weights were similar to those of nitrate fed plants (Fig. 4A). Fig. 4. View largeDownload slide Distribution of plant biomass and 15N-uptake. Plants were cultured hydroponically for 1 week with a continuous supply of a nitrogen-deficient solution. Rapeseed plants were then transferred for 15 d to different treatments at 2 mM-N: CO(15NH2)2 (U), (15NH4)2SO4 (AS), K15NO3 (Nitrate), K15NO3-CO(NH2)2 (NU), KNO3-CO(15NH2)2 (NU), (NH4)2SO4-K15NO3 (AN) and (15NH4)2SO4-KNO3 (AN). (A) Shoot and root dry weights of 15-d-treated plants. (B) Plant nitrogen content. Data represent mean ±standard deviation (n=3) with a bulk of 32 seedlings. Asterisks represent significant differences from the control at*P <0.05, **P <0.01, ***P <0.001 or ns=non significant. Fig. 4. View largeDownload slide Distribution of plant biomass and 15N-uptake. Plants were cultured hydroponically for 1 week with a continuous supply of a nitrogen-deficient solution. Rapeseed plants were then transferred for 15 d to different treatments at 2 mM-N: CO(15NH2)2 (U), (15NH4)2SO4 (AS), K15NO3 (Nitrate), K15NO3-CO(NH2)2 (NU), KNO3-CO(15NH2)2 (NU), (NH4)2SO4-K15NO3 (AN) and (15NH4)2SO4-KNO3 (AN). (A) Shoot and root dry weights of 15-d-treated plants. (B) Plant nitrogen content. Data represent mean ±standard deviation (n=3) with a bulk of 32 seedlings. Asterisks represent significant differences from the control at*P <0.05, **P <0.01, ***P <0.001 or ns=non significant. This difference in growth might reflect differences in N uptake related to N nutrition. The cumulative N uptake was followed by the use of 15N labelling (Fig. 4B). To distinguish nitrogen provided in the mixed nutrient forms (AN and NU), only one element was labelled each time [(NH4)2SO4-K15NO3 or (15NH4)2SO4-KNO3, and K15NO3-CO(NH2)2 or KNO3-CO(15NH2)2; Fig. 4C]. The results revealed the lower capacity of oilseed rape to take up urea (U) when the medium was supplied with this element as the sole N source. Indeed, the cumulative N uptake was about 12-fold lower than for nitrate treatment (from 50.31±2.82 to 4.27±0.24mg 15N plant–1; Fig. 4B). Also, regarding AS, its uptake by plants was about 1.5-fold lower than nitrate. Compared with nitrate-fed plants, those treated with NU or AN, were also characterized by a 22% decrease in 15N content. However, nitrate seems to be preferentially taken up by plants (Fig. 4C). In fact, only 8.6% of the total 15N was provided by urea (3.36±0.19mg 15N plant–1) and 91.4% by nitrate (35.94±2.02mg 15N plant–1) in NU fed plants, while in AN fed plants (Fig. 4C), 45.5% and 54.4% of the total 15N was provided by ammonium (17.81±1.82mg 15N plant–1)and nitrate (21.32±1.42mg 15N plant–1). Furthermore, urea and ammonium uptake were reduced by 45% (from 32.43±3.13 to 17.81±1.82mg 15N plant–1) and 21% (from 4.27±0.24 to 3.36±0.19mg 15N plant–1), respectively, by the presence of nitrate, whose uptake was itself inhibited by 32% in the presence of urea or ammonium (Fig. 4B, 4C). Uptake rate and analysis of BnNRT1.1, BnNRT2.1, BnAMT1.1, and BnDUR3 transcript levels In order to characterize better urea, ammonium, and nitrate transport in plants, the uptake rate was analysed (Fig. 5A) and the relative expression of the genes encoding the urea (BnDUR3), ammonium (BnAMT1.1), and nitrate (BnNRT1.1 and BnNRT2.1) transporters was quantified by qPCR analysis in the roots after 24h and 72h of treatment (Fig. 5B–E). Fig. 5. View largeDownload slide Net uptake rate (A) and relative expression of BnNRT1.1 (B), BnNRT2.1 (C), BnAMT1.1 (D), and BnDUR3 (E). Plants were cultured hydroponically for 1 week with continuous supply of a nitrogen-deficient solution (T=0). Rapeseed plants were then transferred for 24h and 72h to different treatments: CO(15NH2)2(U), (15NH4)2SO4 (AS), K15NO3 (Nitrate), K15NO3-CO(NH2)2 (NU), KNO3-CO(15NH2)2 (NU), (NH4)2SO4-K15NO3 (AN) and (15NH4)2SO4-KNO3 (AN). Data represent mean ±standard deviation (n=3) with a bulk of 32 seedlings. Asterisks represent significant differences from the control at *P <0.05, **P <0.01, ***P <0.001 or ns=not significant. Different superscript letters indicate significant(P <0.05) differences between treatments within each period of treatment. Fig. 5. View largeDownload slide Net uptake rate (A) and relative expression of BnNRT1.1 (B), BnNRT2.1 (C), BnAMT1.1 (D), and BnDUR3 (E). Plants were cultured hydroponically for 1 week with continuous supply of a nitrogen-deficient solution (T=0). Rapeseed plants were then transferred for 24h and 72h to different treatments: CO(15NH2)2(U), (15NH4)2SO4 (AS), K15NO3 (Nitrate), K15NO3-CO(NH2)2 (NU), KNO3-CO(15NH2)2 (NU), (NH4)2SO4-K15NO3 (AN) and (15NH4)2SO4-KNO3 (AN). Data represent mean ±standard deviation (n=3) with a bulk of 32 seedlings. Asterisks represent significant differences from the control at *P <0.05, **P <0.01, ***P <0.001 or ns=not significant. Different superscript letters indicate significant(P <0.05) differences between treatments within each period of treatment. The net uptake rate by roots supplied with urea remained very low throughout the experiment, reaching 0.09 µg 15N h–1 mg–1 root DW after 24h of treatment, and then decreasing to 0.06 µg 15N h–1 mg–1 root DW after 72h (Fig. 5A). Net uptake rates of roots uniformly supplied with NU, AN or nitrate followed the same pattern with time, but during an induction period (24h) the uptake rate reached 2.98, 3.31, and 3.29 µg 15N h–1 mg–1 root DW, respectively, and was then slightly down-regulated during the following 48h to 2.31, 2.50, and 2.80 µg 15N h–1 mg–1 root DW, respectively. No significant differences were found between the AN treatment and Nitrate fed plants (Fig. 5A). A general increase in uptake rate as a function of time, which was slower than NU, AN, and Nitrate, was observed in AS-fed plants, suggesting a lack of down-regulation of ammonium uptake. The relative expression of the BnNRT1.1 (Fig. 5B) and BnNRT2.1 genes (Fig. 5C) was closely related to uptake rates. Indeed, compared with plants grown for 1 week without N (T=0), the results showed a significantly higher expression of BnNRT1.1 (10–16 fold) in the presence of NU, AS, AN, and Nitrate after 24h of treatment, while no change in the relative expression was observed for urea (U)-treated plants (Fig. 5B). After 72h of treatment, BnNRT1.1 expression followed a similar pattern whatever the treatment. The expression of the BnNRT2.1 gene was induced only when nitrate was provided, i.e. in roots of NU (4.5-fold)- and Nitrate (3-fold)-treated plants (Fig. 5C). The same trend was found at 72h, but with lower levels of expression suggesting a down-regulation mostly for AN-treated plants. In the presence of U, AS, and AN, BnNRT2.1 gene expression was maintained in a low steady-state of repression. Regarding ammonium transporters, the results showed a strong down regulation (by 5-fold) of BnAMT1.1 after 24h and 72h, whatever the treatment (Fig. 5D). However, the level of repression was lower after 72h of treatment, except for the AN treatment. BnDUR3 gene expression was maintained at a significantly higher level in roots of U-fed plants, after 24h and 72h of treatment, while it was significantly repressed in the presence of NU, AS, AN, and Nitrate in the growth medium (Fig. 5E). Ammonium assimilation: analysis of BnGDH2 transcript levels The expression in shoots and roots of BnGDH2 was followed after 15 d of treatment (Fig. 6A, 6B) as no significant levels of expression were found between treatments at 24h and 72h (data not shown). In comparison to plants grown for 1 week without N (T=0) or those grown with urea (U), the results showed a slight induction of BnGDH2 in shoots (2.8-fold) and roots (4-fold) of NU-fed plants, while it was strongly induced by AS treatment (6-fold and 30-fold, respectively; Fig. 6A, 6B). A supply in the growth medium of AN or Nitrate has no significant effect on the expression of BnGDH2 in roots (Fig. 6B); despite a slight up-regulation in shoots (Fig. 6A). However, no significant differences were observed between the two treatments. Fig. 6. View largeDownload slide Relative expression of BnGDH2 in shoots (A) and roots (B). Plants were cultured hydroponically for 1 week with continuous supply of a nitrogen-deficient solution (T=0). Rapeseed plants were then transferred for 15 d to different treatments: Urea (U), Ammonium Sulphate (AS), Nitrate, Nitrate Urea (NU), and Ammonium Nitrate (AN). Data represent mean ± standard deviation (n=3) with a bulk of 32 seedlings. Different superscript letters indicate significant (P <0.05) differences between treatments within each period of treatment. Fig. 6. View largeDownload slide Relative expression of BnGDH2 in shoots (A) and roots (B). Plants were cultured hydroponically for 1 week with continuous supply of a nitrogen-deficient solution (T=0). Rapeseed plants were then transferred for 15 d to different treatments: Urea (U), Ammonium Sulphate (AS), Nitrate, Nitrate Urea (NU), and Ammonium Nitrate (AN). Data represent mean ± standard deviation (n=3) with a bulk of 32 seedlings. Different superscript letters indicate significant (P <0.05) differences between treatments within each period of treatment. A signalling role for nitrate: effect of nitrate pulses on urea and ammonium uptake To investigate the possible role of nitrate as a signal and its effect on urea or ammonium uptake, rapeseed seedlings were supplied (at T=0), with U or AS for 24h and 72h. A pulse of 150nM nitrate (KNO3) was added to the nutrient solution every 6h for the duration of the experiment. The cumulative 15N-U and AS uptake was not affected by the nitrate pulse, nor was it affected at 24h or 72h of treatment (Fig. 7A, 7B). However, compared with the plants at t0 (T=0), pulsing U and AS-plants with nitrate strongly induced the root BnNRT1.1 and BnNRT2.1 genes (Fig. 7C, 7D). Indeed, after 24h of treatment, exposures of U and AS-plants to the nitrate pulse induced 10-fold and 15-fold increases in BnNRT1.1, respectively, and 3-fold increases in BnNRT2.1. After 72h of treatment, the relative expression of BnNRT1.1 increased 23-fold in pulsed AS-plants (Fig. 7C), whereas pulsed U-plants increased expression of BnNRT2.1 1.6-fold at the same time (Fig. 7D). The results also showed that the BnDUR3 gene appears to be sensitive to the nitrate signal (Fig. 7E). By comparison, exposing U-plants to a nitrate pulse (U+150nM nitrate) results in a down-regulation of BnDUR3 by 3.5-fold and 1.4-fold after 24h and 72h of treatment, respectively, whereas AS and pulsed AS-plants maintained BnDUR3 expression in a state of repression throughout the duration of the experiment (Fig. 7E). BnAMT1.1 was mostly up-regulated in the starved plants (t0), and then repressed after exposure to the different treatments. However, a nitrate pulse had no effect on BnAMT1.1 gene expression whether plants were grown on U or AS (Fig. 7F). Fig. 7. View largeDownload slide N-urea (A) and N-ammonium (B) uptake, and relative expression of BnNRT1.1 (C), BnNRT2.1 (D), BnDUR3 (E), and BnAMT1.1 (F) in the presence of a pulse of 150nM nitrate (KNO3). Plants were cultured hydroponically for 1 week with continuous supply of a nitrogen-deficient solution (T=0). Rapeseed plants were then transferred for 24h and 72h to different treatments: Urea (U) or Ammonium Sulphate (AS). A pulse of nitrate was added to the nutrient solution every 6h for the duration of the experiment. Data represent mean ±standard deviation (n=3) with a bulk of 32 seedlings. Different superscript letters indicate significant (P <0.05) differences between treatments within each period of treatment. Fig. 7. View largeDownload slide N-urea (A) and N-ammonium (B) uptake, and relative expression of BnNRT1.1 (C), BnNRT2.1 (D), BnDUR3 (E), and BnAMT1.1 (F) in the presence of a pulse of 150nM nitrate (KNO3). Plants were cultured hydroponically for 1 week with continuous supply of a nitrogen-deficient solution (T=0). Rapeseed plants were then transferred for 24h and 72h to different treatments: Urea (U) or Ammonium Sulphate (AS). A pulse of nitrate was added to the nutrient solution every 6h for the duration of the experiment. Data represent mean ±standard deviation (n=3) with a bulk of 32 seedlings. Different superscript letters indicate significant (P <0.05) differences between treatments within each period of treatment. Discussion The use of N fertilizers is essential to keep and/or increase the productivity of cultivated plants. However, excessive N-fertilization may potentially lead to N-losses, and subsequently, lead to economic and environmental impacts. Therefore, to sustain crop production, the fertilizer industry faces a continuing challenge to improve its products, particularly of nitrogenous fertilizers, and to minimize any possible adverse environmental impact. This study aims to highlight the effect of different forms of mineral-fertilizer (urea versus ammonium versus nitrate) and of the use of urease inhibitor, on the physiological efficiency of N, considering specific preferences for certain forms of N, and on the avoidance of N-losses caused by specific N-forms. Experiments were conducted in the field (using lysimeters), to provide information on agronomic aspects and performance of N-fertilization, and laboratory conditions, to identify physiological and molecular mechanisms involved in the nitrogen uptake processes. Yield, N uptake and its distribution in plant organs Plants grown without fertilizer supplementation were characterized by a low yield (–35%) compared with those receiving fertilizers (Fig. 2A). Furthermore, the final yield of 2.3 tonnes ha–1 was recorded whatever the N-form of fertilizers. In Europe, the average seed yield of winter oilseed rape is expected to be between 3 and 4 tonnes ha–1 (Rathke et al., 2006). Hence, the low yields obtained in our experiments are not unusual and may be related to the type of cultivar used (spring oilseed rape), which, compared with winter oilseed rape, shows differences in growth and development. Our results further showed that Fertilizer Use Efficiency (FUE) varies with the nitrogen form (Fig. 2B), and this is closely correlated with N uptake, its partitioning within the plant (Fig. 3A, B) and risk of N volatilization. Urea combined with a urease inhibitor (U+NBPT) shows the best N use efficiency by the plant (46%; Fig. 2B), and it is of interest to note that under the effect of the urease inhibitor, a higher proportion of 15N remained in the soil when plants have been harvested (45.4% compared with 36.6% in the absence of inhibitor; Fig. 3A) which correspond to a more effective immobilization of applied N-fertilizer. Therefore, this could explain the better nitrogen uptake (44.9%; in contrast to 36.4% in the absence of inhibitor, Fig. 3A) and the better seed filling (+ 22%, compared with urea treatment, Fig. 3B). These findings support earlier reports of Basten et al. (2005), which showed an increase of N use efficiency (+7%) of urea+NBPT compared with urea without inhibitor. However, mobilization of N from stems and senescing leaves was lower compared with the urea and AS treatments (Fig. 3B). Indeed, as a result of a lower N availability due to higher volatilization (Fig. 3A), AS treatment was characterized by a lower FUE (29%; Fig. 2B) and hence better mobilization of N from senescing leaves, stems, and taproots to the seeds (Fig. 3B), to achieve a similar seed yield. Thus, AS is not an optimal fertilizer in terms of plant N nutrition when compared with AN, U alone, or combined with the urease inhibitor (U+NBPT). Moreover, it is widely accepted that a combination of nitrate and ammonium (AN treatment) represents the optimum in plant N nutrition and growth (Bloom et al., 1993; Trenkel, 2010). Nitrate promotes organ elongation more than ammonium because of the higher accumulation of osmotic substances and by stimulating cytokinin production (Sakakibara, 2003), whereas ammonium nutrition increases the number of yield components of several crop plants, especially cereals (Camberato and Bock, 1990; Gerendás and Sattelmacher, 1990). On the other hand, considering the potential benefits of urea as a fertilizer (in cost and high N content: 46% N versus 34% for AN) and according to our results, the use of urease inhibitor added to urea may be an interesting option for oilseed rape production. N losses and effect of NBPT Besides the higher FUE and better N recovery of plants, the use of urea as an N-source combined with the urease inhibitor prevented N losses by emission of ammonia (NH3) or dinitrogen oxide (Fig. 3A). It is generally accepted that NH3 volatilization problems result largely from the rapid enzymatic hydrolysis of urea to ammonium carbonate by soil urease (Harrison and Webb, 2001). While these losses may reach 50% of the applied-N (Terman, 1979), our results shows that NBPT significantly reduces (by 68%) the losses of N by volatilization (Fig. 3A), and are in agreement with those observed by Grant et al. (1996) and Sanz-Cobena et al. (2008), who reported a reduction of 36% and 42% in the emitted NH3, respectively. This reduction was associated with the slowdown in urease activity, promoting a decrease of NH4+ availability (Sanz-Cobena et al., 2008). Expressed as a percentage of the N applied, other authors observed, under laboratory conditions, that a reduction in losses can range from 22%, 39% to 51% after fertilizing with a mixture of urea and NBPT (Carmona et al., 1990; Christianson et al., 1990; Antisari et al., 1996). Surprisingly, N-losses with AN treatment are also high (23%), whereas, almost half of the N applied is lost to the atmosphere (44%) when AS was supplied as fertilizer (Fig. 3A). After surface application, NH4+ reaches equilibrium with the dissolved NH3 near the soil surface, increasing the likelihood of gaseous NH3 losses to the atmosphere, particularly when soil pH is high (Table 1; Zhengping et al., 1991; Zaman et al., 2008). N-losses as dinitrogen oxide (N2O) should not be excluded. Zaman et al. (2008) reported that larger amounts of N2O may be emitted from NH4+ and NO3–, as a result of nitrification and denitrification, along with the high moisture content (Fig. 1). Overall, a substantial reduction in N-losses was achieved by NBPT-treated urea. This result suggests that the uses of the urease inhibitor could be environmentally and economically useful amendments to urea-based fertilizers. Net nitrate, ammonium, and urea uptake under controlled conditions and the effect of urea and ammonium on growth Our results showed that strict ammonium (AS treatment) and urea (U treatment) causes a decrease (50% and 75%, respectively) in oilseed rape growth when compared with strict nitrate nutrition, after 15 d from the onset of the treatments (Fig. 4A). These results were in agreement with those reported by other authors in various plant species (Bradley et al., 1989; Tan et al., 2000; Houdusse et al., 2005; Merigout et al., 2008; Garnica et al., 2009). Furthermore, this negative effect was correlated with a reduced capacity for ammonium and urea uptake by plants (–35% and –90%, respectively; Fig. 4B). Hence, it appears that the deleterious effects on growth associated with urea may differ, at a physiological level, from those associated with ammonium. The latter may have toxic effects, while urea-treated plants seem to suffer from nitrogen deficiency. Indeed, it is widely accepted that, in most higher plants, excessive ammonium is toxic (Britto and Kronzucker, 2002). This fact has been related to the accumulation of NH4+ in plant tissues, leading to its rapid assimilation into the amino acid glutamate by the glutamine synthetase/glutamate synthase pathway (Lea and Miflin, 1974). At the site of NH4+ uptake rate (AS treatment; Fig. 5A), the results showed lower ammonium uptake rates after 24h and 72h of treatment, when compared with nitrate-fed plants. This is correlated with the down-regulation of BnAMT1.1 in the roots when plants were shifted from nitrogen-deficient to nitrogen-sufficient nitrogen conditions (Fig, 5D). AtAMT1.1 encodes for the major ammonium high-affinity transport system, and it is generally repressed under nitrogen sufficiency (Yuan et al., 2007). Hence, a rapid decrease of its relative expression (Fig. 5D) may lead to a decrease in ammonium uptake capacities to avoid cellular toxicity (Britto and Kronzucker, 2002). GDH may play a major role in keeping the NH4+ levels in the cytoplasm below toxicity (Tercé-Laforgue et al., 2004), and could operate via amination. It has been established in vivo that GDH1 solely deaminates glutamate (Purnell and Botella, 2007), while GDH2 exhibits strong deaminating activity and only very low aminating activity (Skopelitis et al., 2007). Recently, a transcriptomic analysis of Arabidopsis roots supplied with nitrate or ammonium showed that GDH2 was specifically induced by ammonium (Patterson et al., 2010). Correspondingly, in the long term (15 d), a strong up-regulation of root and shoot BnGDH2 was observed in AS-treated plants (30-fold and 6-fold, respectively; Fig. 6A, B). Taken together, we hypothesize that the induction of this gene is a response to the externally provided NH4+, preventing its accumulation. The higher expression of BnGDH2 in roots (by 5-fold) than in shoots suggests that the roots are the main NH4+ assimilatory organ in oilseed rape. By comparison with the nitrate treatment, plants grown on AS are characterized by root growth inhibition (Fig. 4A). Li et al. (2010) showed that NH4+ supplied to roots in Arabidopsis affects cell elongation, and that growth inhibition was associated with NH4+ efflux in the elongation root zone. Other studies have also proposed that the auxin signal is involved in NH4+ toxicity leading to growth inhibition (Cao et al., 1993). Thus, induction of BnNRT1.1 by AS (Fig. 5B), which was unexpected at first glance, makes an interesting parallel with the recent demonstration of auxin influx facilitation by NRT1.1 (Krouk et al., 2010). As for ammonium-fed plants, it has been reported that nitrogen nutrition based on urea leads to reduced growth because of its accumulation in toxic amounts, causing subsequently leaf-tip necrosis (Krogmeier et al., 1989). Also, compared with plants receiving nitrate or ammonium-nitrate, plants receiving urea as the sole N source are characterized by symptoms of N-starvation (Merigout et al., 2008). The low N-urea content in the plant (Fig. 4B) and a drastic reduction of its uptake rate (Fig. 5A), confirms the hypothesis of a nitrogen deficiency in oilseed rape. The molecular reason for the inefficient use of urea by plants remains unclear but the extremely low urea uptake rate that was measured may be the reason. However, analysis of BnDUR3 expression in roots gave some interesting results with regard to urea uptake (Fig. 5E). In a similar way to that of AtAMT1.1, BnDUR3 was expressed at a high level in N-deficient roots (Fig. 5E). Because it has been shown that re-supply of urea to nitrogen-deficient roots strongly increased the level of BnDUR3 transcripts, suggesting a substrate-inducible transport system (Kojima et al., 2007), it cannot be excluded that under our conditions, BnDUR3 expression was induced by the endogenous urea accumulation that occurred in N-starved plants. Otherwise, this gene tends to retain the same pattern of expression as under nitrogen-deficient conditions when plants were re-supplied with urea for 72h (Fig. 5E). Hence, having observed a very low urea uptake rate (Fig. 5A), it is suggested that such gene expression patterns indicated that BnDUR3 is a component of the stress response to nitrogen-deficiency in rapeseed. The low-affinity transport systems may also contribute to ammonium or urea uptake since N was supplied in the millimolar range. Future characterizations of these transport systems would be of great interest. Nitrate corrects the negative effect of urea- and ammonium-fed plants Many studies have reported that the negative effects associated with NH4+ and urea nutrition are corrected by the presence of nitrate in the growth medium (Britto and Kronzucker, 2002; Garnica et al., 2009). Compared with ammonium- and urea-fed plants, the presence of nitrate along with NH4+ (AN) and urea (NU) increases the N-uptake by 1.2-fold and 9-fold, respectively (Fig. 4B), and therefore restores the growth of oilseed rape (Fig. 4A). However, enhancement of NH4+ and urea uptake by NO3– was not observed (Fig. 4C). This finding is contrary to the results reported by Garnica et al. (2009), who showed a significant enhancement of NH4+ and urea uptake in wheat by NO3– supply. Indeed, in such mixed strategies of nutrition (AN and NU), oilseed rape preferentially takes up NO3–, and this is shown especially in the case of NU treatment, where 91% of N is derived from nitrate, in contrast to 54% in the case of AN treatment (Fig. 4C). This may be partly related to the nitrophilic character of Brassicaceae. Lainé et al. (1993) reported that the peak rate of nitrate uptake is higher in many Brassicaceae than in many other crop plants (e.g. members of the Poaceae and Legumineae). Also, these results are closely correlated with the expression pattern of BnNRT1.1 and BnNRT2.1 genes, which are significantly up-regulated in AN- and NU-fed plants (Fig. 5B, C), and especially in the case of BnNRT1.1 (Fig. 5B). On the other hand, we also observed that the presence of NH4+ or urea greatly delayed NO3– uptake (Fig. 4B, 4C). This result was expected, since it has been described by many authors in various plant species (Criddle et al., 1988; Kronzucker et al., 1999; Garnica et al., 2009), and this could be attributed to the down-regulation of BnNRT2.1 by urea and ammonium (Fig. 5C), and BnNRT1.1 by urea only (Fig. 5B). In summary, the results indicate that the beneficial effect of nitrate in the development of plants fed with ammonium or urea seems to be related to the nutritional character of nitrate. The increase in root growth observed in the mixed nutrition should also be a specific effect of nitrate, which involves the NRT1.1 gene (Fig. 5B; Krouk et al., 2010). However, effects on the flow of ammonium and urea assimilation should not be excluded. Nitrate as a signal molecule Nitrate is not only the main nitrogen source for plant nutrition but it also plays a major role as a signal molecule modulating plant metabolism and growth (Crawford, 1995). Our results showed a strong but expected induction of the BnNRT1.1 and BnNRT2.1 genes when ammonium (AS) and urea (U)-plants received NO3– pulses (Fig. 7C, 7D), confirming the efficiency of the low concentration of nitrate provided as a pulse (150nM). NRT1.1 is considered as a dual affinity nitrate transporter, belonging to both the HATS and LATS (Liu et al., 1999) and dual affinity nitrate sensor, depending on its phosphorylation (Liu and Tsay, 2003). Therefore, Ho et al. (2009) demonstrated that the sensing function of NRT1.1 may be separate from its NO3– transport activity. Indeed, it has been established that NRT1.1 is involved not only in transporting nitrate, but also facilitates uptake of auxin, and favours its basipetal transport in lateral roots, at low nitrate availability (Krouk et al., 2010). Surprisingly, BnDUR3 expression also seems to be sensitive to extremely low concentrations of NO3– (Fig. 7E). This suggests another additional role for nitrate as a signal. Our results show no effect of nitrate pulsing on ammonium or urea uptake (Fig. 7A, B). From controlled conditions to the field: anysignificant uptake of urea? Such a differential in the experimental designs used in this study (hydroponics versus field lysimeter), provides results that, at first glance, appear to have possible discrepancies. Indeed, results from the hydroponic experiments provide elements of knowledge on urea uptake without soil and/or microorganism interference. On the other hand, the use of hydroponic devices with the isotopic labelling of urea give a gross access to N uptake whatever the form taken up resulting from biotic or abiotic transformations. Thus, from the hydroponic experiments it can be postulated that urea uptake does not contribute significantly to the N nutrition of a nitrophilic species such as Brassica napus L. because of an extremely low uptake rate potential, which triggers symptoms of N deficiency for urea-fed plants (reduced growth, up-regulation of AMT1 and DUR3 genes). However, urea fertilization in the field combined with a urease inhibitor provide the best FUE with the lowest N volatilization, which means that microbial hydrolysis of urea, even when reduced, is sufficient to deliver nitrate and ammonium for plant N uptake, as it can be postulated that urea itself is not significantly taken up, and contributes, at least indirectly, to the N nutrition of oilseed rape. It has been shown that the supply of urease inhibitor may have phytotoxic effects, since NBPT (Cruchaga et al., 2011) or phenylphosphorodiamidate (PPD; Arkoun et al., 2012) can interact with plant metabolism. However, it must be pointed out that these previous works were conducted in hydroponic conditions under which the contact of the roots with the urease inhibitor was permanent and with fairly high and constant concentrations. However, in the presence of soil, this effect may be only transitory (Artola et al., 2011), and under our lysimeter conditions, the benefits of NBPT-reducing ammonia volatilization and increasing N-urea uptake would appear to far outweigh any observed short-term modification in nitrogen metabolism. Conclusion The contribution of different mineral N fertilizers to yield or N-use efficiency of oilseed rape is of special importance. Overall, ammonium sulphate fertilization showed the worst effects, with a reduction in growth and increased volatilization leading to lower N availability while nitrate ammonium or urea fertilization provided improvements in these parameters. The addition of NBPT with urea, a widely used urease inhibitor, restricted N volatilization and, as a consequence, promoted a higher soil immobilization. However, in hydroponic conditions, urea-grown plants showed a strong reduction in their growth compared with nitrate supply alone, and this reduction was higher than with ammonium sulphate. This could be due to N deficiency effects resulting from very low urea uptake rates rather than to urea or ammonium derived toxicity per se as illustrated by the fact that BnGDH2 (potentially involved in ammonium detoxification), which was strongly up-regulated with ammonium sulphate nutrition, was not affected in urea-grown plants. Moreover, the expression of both BnAMT1.1 and BnDUR3, encoding ammonium and urea transporters, was up-regulated by N starvation as well as by urea nutrition, the latter being sensitive to nitrate, which acts as a signal to BnNRT1.1 and BnNRT2.1 expression in a similar way. Overall, the results suggest that urea is not taken up in the field at a significant level, even in the presence of a urease inhibitor, however, this does not negatively affect the improved fertilizer use efficiency resulting from delayed microbial hydrolysis of urea. Table 1 Physical and chemical properties of the soil lysimeters Depth
(cm)  Particle size distribution (%)  pH
 (water)  Organic C
(mg C g–1)  Total N
(mg N g–1)  C/N  CEC
(cmol kg–1)  Total
CaCO3
 (%)  Sand  Silt  Clay    0–20  32  38  30  7.9  25.4  3.21  7.9  16.5  10  20–40  34  38  28  8.1  15  1.87  8  11.5  12  40–65  41  35  24  8.3  7.6  0.93  8.2  7.2  28  65–100  49  34  17  8.4  3.4  0.4  8.5  4.6  38  Depth
(cm)  Particle size distribution (%)  pH
 (water)  Organic C
(mg C g–1)  Total N
(mg N g–1)  C/N  CEC
(cmol kg–1)  Total
CaCO3
 (%)  Sand  Silt  Clay    0–20  32  38  30  7.9  25.4  3.21  7.9  16.5  10  20–40  34  38  28  8.1  15  1.87  8  11.5  12  40–65  41  35  24  8.3  7.6  0.93  8.2  7.2  28  65–100  49  34  17  8.4  3.4  0.4  8.5  4.6  38  View Large Acknowledgements Authors acknowledge Marie-Paule Bataillé and Raphaël Ségura for IRMS analyses, Dominique Ballois for helping with plant culture and harvests, and Laurence Cantrill for kindly improving the English of the manuscript. The authors thank the ‘Pôle de compétitivité Mer-Bretagne’ and the FUI (Fond Unique Interministériel from the French government) which supported this work conducted through the AZOSTIMER project. References Antisari LV Marzadori C Gioacchini P Ricci S Gessa C 1996 Effects of the urease inhibitor N-(n-butyl) phosphorothioic triamide in low concentrations on ammonia volatilization and evolution of mineral nitrogen. Biology and Fertility of Soils  22 196– 201 Google Scholar CrossRef Search ADS   Arkoun M Jannin L Laîné P Etienne P Masclaux-Daubresse C Citerne S Garnica M Garcia-Mina JM Yvin JC Ourry A 2012 A physiological and molecular study of the effects of nickel deficiency and phenylphosphorodiamidate (PPD) application on urea metabolism in oilseed rape (Brassica napusL.). Plant and Soil 10.1007/s11104-012-1227-2. Artola E Cruchaga S Ariz I Moran JF Garnica M Houdusse F Garcia Mina JM Irigoyen I Lasa B Aparicio-Tejo PM 2011 Effect of N-(n-butyl) thiophosphoric triamide on urea metabolism and the assimilation of ammonium by Triticum aestivumL. Plant Growth Regulation  63 73– 79 Google Scholar CrossRef Search ADS   Basten M Brynildsen P Belzen R 2005 Stabilized urea for enhanced nitrogen use efficiency. IFA International Workshop on Enhanced-Efficiency Fertilizers, Frankfurt. International Fertilizer Industry Association (IFA) Paris, France Bloom AJ Jackson LE Smart DR 1993 Root growth as a function of ammonium and nitrate in the root zone. Plant, Cell and Environment  16 199– 206 Google Scholar CrossRef Search ADS   Bradley DP Morgan MA O’Toole P 1989 Uptake and apparent utilization of urea and ammonium nitrate in wheat seedlings. Fertilizer Research  20 41– 49 Google Scholar CrossRef Search ADS   Bremner JM Chai HS 1989 Effects of phosphoroamides on ammonia volatilization and nitrite accumulation in soils treated with urea. Biology and Fertility of Soils  8 227– 230 Britto DT Kronzucker HJ 2002. NH4+ toxicity in higher plants: a critical review. Journal of Plant Physiology   159 567– 584 Google Scholar CrossRef Search ADS   Camberato JJ Bock BR 1990 Spring wheat response to enhanced ammonium supply. I. Dry matter and nitrogen content Agronomy Journals  82 463– 467 Google Scholar CrossRef Search ADS   Cao YW Glass ADM Crawford NM 1993 Ammonium inhibition of Arabidopsisroot growth can be reversed by potassium and auxin resistance mutations aux1, axr1and axr2. Plant Physiology  102 983– 989 Google Scholar CrossRef Search ADS PubMed  Carmona G Christianson CB Byrnes BH 1990 Temperature and low concentration effects of the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) on ammonia volatilisation from urea. Soil Biology and Biochemestery  22 933– 937 Google Scholar CrossRef Search ADS   Cerezo M Tillard P Filleur S Munos S Daniel-Vedele F Gojon A 2001 Major alterations of the regulation of root NO3– uptake are associated with the mutation of NRT2.1 and NRT2.2 genes in Arabidopsis Plant Physiology  127 262– 271 Google Scholar CrossRef Search ADS PubMed  Christianson CB Byrnes BH Carmona G 1990 A comparison of the sulfur and oxygen analogs of phosphoric triamide urease inhibitors in reducing urea hydrolysis and ammonia volatilization. Fertilizer Research  26 21– 27 Google Scholar CrossRef Search ADS   Crawford NM 1995 Nitrate: nutrient and signal for plant growth. The Plant Cell  7 859– 868 Google Scholar CrossRef Search ADS PubMed  Crawford NM Glass ADM 1998 Molecular and physiological aspects of nitrate uptake in plants Trends in Plant Science  3 389– 395 Google Scholar CrossRef Search ADS   Criddle R Ward M Huffaker R 1988 Nitrogen uptake by wheat seedlings, interactive effects of four nitrogen sources: NO3–, NO2–, NH4+, and urea. Plant Physiology   86 166– 175 Google Scholar CrossRef Search ADS PubMed  Cruchaga S Artola E Lasa B Ariz I Irigoyen I Moran JF Aparicio-Tejo PM 2011 Short term physiological implications of NBPT application on the N metabolism of Pisum sativumand Spinacea oleracea. Journal of Plant Physiology  168 329– 336 Google Scholar CrossRef Search ADS PubMed  Cruz C Castillo M Domίnguez CN Juanarena N Aparicio-Tejo P Lamsfus C 2003 The importance of nitrate signalling in plant ammonium tolerance: spinach as a case study.In: Actas XV Reunion de la Sociedad Española de Fisiologίa Vegetal y VIII Congreso Hispano-Luso  Palma de Mallorca: Sociedad Española de Fisiologίa Vegetal Forde BG 2000 Nitrate transporters in plants: structure, function and regulation. Biochimica et Biophysica Acta  1465 219– 235 Google Scholar CrossRef Search ADS PubMed  Garnica M Houdusse F Yvin JC Garcia-Mina JM 2009 Nitrate modifies urea root uptake and assimilation in wheat seedlings. Journal of the Science of Food and Agriculture  89 55– 62 Google Scholar CrossRef Search ADS   Gazzarrini S Lejay L Gojon A Ninnemann O Frommer WB von Wirén N 1999 Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsisroots. The Plant Cell  11 937– 947 Google Scholar CrossRef Search ADS PubMed  Gerendàs J Sattelmacher B 1990 Influence of nitrogen form and concentration on growth and ionic balance of tomato (Lycopersicum esculentum) and potato (Solanum tuberosum).In: Van Beusichem ML , ed. Plant nutrition-physiology and applications  Kluwer Academic Publishers 33– 37 Gill JS Bijay-Singh Khind CS Yadvinder-Singh 1997 Efficiency of N-(n-butyl) thiophosphoric triamide in retarding hydrolysis of urea and ammonia volatilization losses in a flooded sandy loam soil amended with organic materials. Nutrient Cycling in Agroecosystems  53 203– 207 Google Scholar Grant CA Jia S Brown KR Bailey LD 1996 Volatile losses of NH3 from surface-applied urea and urea ammonium nitrate with and without the urease inhibitors NBPT or ammonium thiosulphate. Canadian Journal of Soil Science  76 417– 419 Google Scholar CrossRef Search ADS   Harrison R Webb J 2001 A review of the effect of N fertilizer type on gaseous emissions. Advances in Agronomy  73 65– 108 Ho CH Lin SH Hu HC Tsay YF 2009 CHL1 functions as a nitrate sensor in plants. Cell  138 1184– 1194 Google Scholar CrossRef Search ADS PubMed  Hofman G Van Cleemput O 2004 Soil and plant nitrogen  International fertilizer industry association (IFA) Paris, France Houdusse F Zamarreno AM Garnica M Garcia-Mina JM 2005 The importance of nitrate in ameliorating the effects of ammonium and urea nutrition on plant development: the relationships with free polyamines and proline plant contents. Functional Plant Biology  32 1057– 1067 Google Scholar CrossRef Search ADS   Kaiser BN Rawat SR Siddiqi YM Masle J Glass ADM 2002 Functional analysis of an Arabidopsis T-DNA ‘knockout’ of the high-affinity NH4+ transporter AtAMT1.1. Plant Physiology   130 1263– 1275 Google Scholar CrossRef Search ADS PubMed  Kojima S Bohner A Gassert B Yuan L von Wirén N 2007 AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient Arabidopsisroots. The Plant Journal  52 30– 40 Google Scholar CrossRef Search ADS PubMed  Krogmeier MJ McCarty GW Bremner JM 1989 Potential phytotoxicity associated with the use of soil urease inhibitors. Proceedings of the National Academy of Sciences, USA  86 1110– 1112 Google Scholar CrossRef Search ADS   Kronzucker HJ Glass ADM Siddiqi MY 1999 Inhibition of nitrate uptake by ammonium in barley. Analysis of component fluxes. Plant Physiology  120 283– 291 Google Scholar CrossRef Search ADS PubMed  Krouk G Lacombe B Bielach A et al.   2010 Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Developmental Cell  18 927– 937 Google Scholar CrossRef Search ADS PubMed  Google Scholar Lainé P Ourry A Macduff J.H Boucaud J Salette J 1993 Kinetic parameters of nitrate uptake by different catch crop species: effects of low temperatures or previous nitrate starvation. Physiologia Plantarum  88 85– 92 Google Scholar CrossRef Search ADS   Lea PJ Miflin BJ 1974 Alternative route for nitrogen assimilation in higher plants. Nature   251 516– 614 Google Scholar CrossRef Search ADS   Li Q Li BH Kronzucker HJ Shi WM 2010 Root growth inhibition by NH4+ in Arabidopsisis mediated by the root tip and is linked to NH4+ efflux and GMPase activity. Plant, Cell and Environment  33 1529– 1542 Google Scholar CrossRef Search ADS   Liu KH Huang CY Tsay YF 1999 CHL1 is a dual-affinity nitrate transporter of Arabidopsis involving multiple phases of nitrate uptake. The Plant Cell  11 865– 874 Google Scholar CrossRef Search ADS PubMed  Liu KH Tsay YF 2003 Switching between the two action modes of the dual affinity nitrate transporter CHL1 by phosphorylation. EMBO Journal  22 1005– 1013 Google Scholar CrossRef Search ADS PubMed  Liu LH Ludewig U Frommer WB von Wirén N 2003 AtDUR3 encodes a new type of high-affinity urea/H+ symporter in Arabidopsis. The Plant Cell  15 790– 800 Google Scholar CrossRef Search ADS PubMed  Livak KJ Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods  25 402– 408 Google Scholar CrossRef Search ADS PubMed  Malhi SS Grant CA Johnston AM Gill KS 2001 Nitrogen fertilization management for no till cereal production in the Canadian Great Plains: a review. Soil Tillage Research  60 101– 122 Google Scholar CrossRef Search ADS   Malhi SS Nyborg M 1991 Recovery of 15N-labelled urea: influence of zero tillage, and time and method of application. Fertilizer Research  28 263– 269 Google Scholar CrossRef Search ADS   Marschner H 1995 Functions of mineral nutrition: macronutrients.In: Marschner H , ed. Mineral nutrition of higher plants  London: Academic Press Mérigout P Lelandais M Bitton F Renou JP Briand X Meyer C Daniel-Vedele F 2008 Physiological and transcriptomic aspects of urea uptake and assimilation in Arabidopsisplants. Plant Physiology  147 1225– 1238 Google Scholar CrossRef Search ADS PubMed  Ninnemann O Jauniaux JC Frommer WB 1994 Identification of a high affinity NH4+ transporter from plants. EMBO Journal  13 3464– 3471 Google Scholar PubMed  Patterson K Cakmak T Cooper A Lager I Rasmusson AG Escobar MA 2010 Distinct signalling pathways and transcriptome response signatures differentiate ammonium- and nitrate-supplied plants. Plant, Cell and Environment  33 1486– 1501. Pearson JN Finnemann J Schjoerring JK 2002 Regulation of the high-affinity ammonium transporter (BnAMT1.2) in the leaves of Brassica napusby nitrogen status Plant Molecular and Biology  49 483– 490 Google Scholar CrossRef Search ADS   Phongpan S Freney JR Keerthisinge DG Chaiwanakupt P 1995 Use of phenylphosphorodiamidate and N-(n-butyl)thiophosphoric triamide to reduce ammonia loss and increase grain yield following application of urea to flooded rice. Fertilizer Research   41 59– 66 Google Scholar CrossRef Search ADS   Purnell MP Botella JR 2007 Tobacco isoenzyme 1 of NAD(H)-dependent glutamate dehydrogenase catabolizes glutamate in vivo. Plant Physiology  143 530– 539 Google Scholar CrossRef Search ADS PubMed  Rathke GW Behrens T Diepenbrock W 2006 Integrated nitrogen management strategies to improve seed yield, oil content and nitrogen efficiency of winter oilseed rape (Brassica napusL.): a review. Agriculture, Ecosystems and Environment  117 80– 108 Google Scholar CrossRef Search ADS   Rhodes D Brunk OG Magalhaes JR 1989 Assimilation of ammonium by glutamate dehydrogenase?In: Poulten TE Romeo JT Conn EE , eds. Recent advances in phytochemistry  New York: Plenum Press 191– 226 Sakakibara H 2003 Nitrate-specific and cytokinin-mediated nitrogen signaling pathways in plants. Journal of Plant Research  116 253– 257 Google Scholar CrossRef Search ADS PubMed  Sanz-Cobena A Misselbrook TH Arce A Mingot JI Diez JA Vallejo A 2008 An inhibitor of urease activity effectively reduces ammonia emissions from soil treated with urea under Mediterranean conditions. Agriculture, Ecosystems and Environment  126 243– 249 Google Scholar CrossRef Search ADS   Skopelitis DS Paranychiankis NV Kouvarakis A Spyros A Stephanou EG Roubelakis-Angelakis KA 2007 The isoenzyme 7 of tobacco NAD(H)-dependent glutamate dehydrogenase exhibits high deaminating and low aminating activity. Plant Physiology  145 1726– 1734 Google Scholar CrossRef Search ADS PubMed  Sohlenkamp C Shelden M Howitt S Udvardi M 2000 Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants. FEBS Letters  467 273– 278 Google Scholar CrossRef Search ADS PubMed  Sommer SG Hutchings NJ 2001 Ammonia emissions from field applied manure and its reduction. European Journal of Agronomy  15 1– 15 Google Scholar CrossRef Search ADS   Tan XW Ikeda H Oda M 2000 The absorption, translocation, and assimilation of urea, nitrate and ammonium in tomato plants at different plant growth stages in hydroponic culture. Scientia Horticultura  84 275– 283. Google Scholar CrossRef Search ADS   Tercé-Laforgue T Dubois F Ferrario-Méry S Pou de Crecenzo MA Sangwan R Hirel B 2004 Glutamate dehydrogenase of tobacco (Nicotiana tabacumL.) is mainlyinduced in the cytosol of phloem companion cells whenammonia is provided either externally or released during photorespiration Plant Physiology  136 4308– 4317 Google Scholar CrossRef Search ADS PubMed  Terman GL 1979 Volatilization losses of nitrogen as ammonia from surface-applied fertilizers, organic amendments, and crop residues. Advances in Agronomy  31 189– 223 Trenkel ME 2010 Slow- and controled-release and stabilized fertilizers. An option for enhancing nutrient use efficiency in agriculture  Paris, France: International fertilizer industry association (IFA) Vlek PLG Carter MF 1983 The effect of soil environment and fertilizer modifications on the rate of urea hydrolysis. Soil Science  136 56– 63 Google Scholar CrossRef Search ADS   Watson CJ Miller H Poland P Kilpatrick DJ Allen MDB Garrett MK Christianson CB 1994 Soil properties and the ability of the urease inhibitor N-(n-butyl) thiophosphoric triamide (nBTPT) to reduce ammonia volatilization from surface-applied urea. Soil Biology and Biochemistry   26 1165– 1171 Google Scholar CrossRef Search ADS   Yamaya T Oaks A Rhodes D Matsumoto H 1986 Synthesis of 15N by mitochondria isolated from pea and corn shoots. Plant Physiology  81 754– 757 Google Scholar CrossRef Search ADS PubMed  Yuan L Loqué D Ye F Frommer WB von Wirèn N 2007 Nitrogen-dependent posttranscriptional regulation of the ammonium transporter AtAMT1;1. Plant Physiology  143 732– 744 Google Scholar CrossRef Search ADS PubMed  Zaman M Nguyen ML Blennerhassett JD Quin BF 2008 Reducing NH3, N2O and NO3– -N losses from a pasture soil with urease or nitrification inhibitors and elemental S-amended nitrogenous fertilizers. Biology and Fertility of Soils  44 693– 705 Google Scholar CrossRef Search ADS   Zhengping W Van Cleemput O Liantie L Baert L 1991 Effect urease inhibitors on urea hydrolysis and ammonia volatilization. Biology and Fertility of Soils  11 43– 47 Google Scholar CrossRef Search ADS   © The Author [2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
TI - Hydroponics versus field lysimeter studies of urea, ammonium and nitrate uptake by oilseed rape(Brassica napus L.)
JF - Journal of Experimental Botany
DO - 10.1093/jxb/ers183
DA - 2012-08-29
UR - https://www.deepdyve.com/lp/oxford-university-press/hydroponics-versus-field-lysimeter-studies-of-urea-ammonium-and-PN13dRyE6M
SP - 5245
EP - 5258
VL - 63
IS - 14
DP - DeepDyve
ER -