Short running title: NBPT implications on nitrogenmetabolism
Corresponding author: Pedro M. Aparicio-Tejo
Mailing address: Institute of Agri-biotechnology Institute (IdAB). UPNa-CSIC-GN. 31192 Mutilva Baja. Navarra. Spain.
Phone: (+34)948168000.
Fax: (+34)948168930
E-mail:
Short term physiological implications of NBPT application on the N metabolism of Pisum sativum and Spinacea oleracea
Saioa Cruchagaa, Ekhiñe Artolaa, Berta Lasaa, Idoia Arizb, Ignacio Irigoyenc, Jose Fernando Moranb, Pedro M. Aparicio-Tejob,*
aDpto. Ciencias del Medio Natural. Campus Arrosadía. PublicUniversity of Navarra. 31600 Pamplona. Navarra. Spain
bInstitute of Agri-biotechnology Institute (IdAB). UPNa-CSIC-GN. 31192 Mutilva Baja. Navarra. Spain
cDpto. Producción Agraria. Campus Arrosadía. PublicUniversity of Navarra. 31600 Pamplona. Navarra. Spain
ABSTRACT
The application of urease inhibitors in conjunction with urea fertilizers as a means of reducing N losses 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 soleN source. After two weeks of growth for pea, and three 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 NBPTcontent in these tissues was determined 48 hours after inhibitor application. The results suggest that the effect of NBPT on spinach and pea urease activity is different, with peabeing most affected by this treatment, and that the NBPT absorbed by the plant causes a clear inhibition of the urease activity in pea leaf and root. The high urea concentration observed in leaves is associated with the development of necrotic leaf margins and is further evidence of NBPT inhibition in these plants. A decrease in the ammonium content in root, where N assimilation mainly takes place, was also observed. Consequently, total amino acid contents were drastically reduced upon NBPT treatment, thus 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 activityin this species.
Keywords: ammonium, N-(n-butyl) thiophosphoric triamide, NBPT, urease inhibitor, urea
Abbreviations: NBPT, (N-(n-butyl) thiophosphoric triamide)
Introduction
Urease, the only Ni-dependent metalloenzyme in eukaryotes, catalyzes the hydrolysis of urea to ammonium and carbon dioxide, thereby allowingthese organisms to use external or internally generated urea as an N source (Andrews et al., 1984; Mobley and Hausinger, 1989; Mobley et al., 1995). In plants, urea is mainly derived from arginine (Polacco and Holland, 1993), although it can also be generated by ureide catabolism (Todd and Polacco, 2004; Muñoz et al., 2006). Plants are able to utilize urea applied to foliage (Leacox and Syvertsen, 1995) or they can take it up through the roots as a whole molecule, as demonstrated by hydroponic studies (Harper, 1984).
It is well known that the rapid hydrolysis of urea-based fertilizers by bacterial ureases in the soil results in substantial N losses due to ammonia volatilization. Indeed, it has been estimated that more than 50 % of the N fertilizer applied is lost in this way (Terman, 1979). One approach to improving the efficiency of urea application is to combine it with urease inhibitors, whichdelay the hydrolysis process and thereby extend urea availability by avoiding nitrate leaching andreducing NH3 loss. Among the various types of urease inhibitors which have been identified and tested, N-(n-butyl) thiophosphoric triamide (NBPT) has proved to be significantly effective at relatively low concentrations under laboratory conditions (Gill et al., 1999).
NBPT shows similar solubility and diffusivity characteristics to urea (Carmona et al., 1990), and its application in conjunction with urea can affect plant urease activity and cause some leaf-tip scorch, although these effects are transient and short-lived (Watson and Miller, 1996). When urease activity is low due to inadequate Ni supply or urease inhibitor application, urea may accumulate to considerable levels, particularly in urea-treated plants (Gerendás and Sattelmacher, 1997). This accumulation, as well as some physiological effects and the disruption of amino acid metabolism, has been described in wheat, soybean, sunflower, ryegrass and pecan (Watson and Miller, 1996; Gerendás and Sattelmacher, 1997; Bai et al., 2006). Previous studies from our group found inter-specific differences in ammonium sensitivity that seemed to be related to differences in the organ where ammonium is assimilated, as well as to the assimilation pathway (Lasa et al. 2002). In the current study, the short-term physiological implications of NBPT application for N metabolism in pea and spinach plants were investigated.
Material and methods
Plant growth conditions and experimental set-up
Pea (Pisum sativum L., “Snap-pea”) and spinach (Spinacea oleracea L., “Gigante de invierno”) seeds were sown in vermiculite:perlite (2:1) and irrigated with distilled water. Pea seeds were previously surface sterilized as described by Labhilili et al. (1995). After ten days, seedlings were transplanted to a continuously aerated hydroponic culture with eight seedlings/8-L tank. The nutrient solution used was that described by Rigaud and Puppo (1975) (N-freesolution) supplemented with urea (5 mM for pea and 1.5 mM for spinach) as previous studies have shown that these concentrations are optimal for a maximum growth of each species. The hydroponic solution was changed every seven days during the first two weeks for pea and three weeks for spinach. After that time, treated plants were supplemented with NBPT at a final concentration of 100 µM. Urea isotopically labelled with 15N (5%) was applied in the last solution change just before the application of NBPT. Hydroponically cultured plants were grown under controlled conditions at 22/18 ºC (day/night), 60/80 % relative humidity, 16/8 h photoperiod and 600 µmol m-2 s-1 photosynthetic photon flux. Plant material from leaves and roots was collected at days 0, 1, 2, 4, 7 and 9 after treatment initiation, frozen in liquid N2 and stored at -80 ºC. Younger pea leaves in theirearly stages of development were also taken separately at day 9. Dry material was obtained by drying in an oven at 80 °C for 48 h.
NBPT determination
NBPT was analyzed by HPLC-ESI-MS. The instrument consisted of an Agilent series 1100 chromatograph system and an ion trap SL model spectrometer. Extraction was carried out from frozen tissues in distilled water and the supernatant obtained after centrifugation was used.
Separation was performed on an HPLC column (2.1x30 mm; 3.5 µm, Zorbax SB-C18) at 25 ºC. The mobile phase was 40:60 distilled water + 0.1% formic acid:methanol + 0.1 % formic acid (flow rate: 0.1 mL/min).
All analyses were performed using the ESI interface with the following settings: positive ionisation mode; 40 psi of nebulizer pressure, nitrogen flow of 8L/min and 350ºC. MS/MS spectra of ions were obtained by collision-induced dissociation in the ion trap with helium. Quantification was based on the 151 and 74 mass ions generated from the 168 ion precursor [M+H]+.
Determination of urease activity
Urease was extracted from frozen plant material in 50 mM phosphate buffer (pH 7.5) containing 50 mM NaCl and 1 mM EDTA. In-Gel detection of urease activity was performed following the methodology described by Witte and Medina-Escobar (2001) using jack bean urease (Sigma EC 3.5.1.5) as standard.
Determination of urea content
The urea concentration was determined using the method described by Witte et al. (2002). In order to avoid interference from other molecules, such as ammonium and some amino acids, the extracts were previously passed through ion-exchange columns (sample extraction products; Water Oasis; MCX and MAX), with 900 µL of the reagent described by Kyllingsbæk (1975) being added to 300 µL of extract.
Quantification of ammonium and protein content
Ammonium was extracted from frozen tissue by treatment with water at 80 °C for 5 minutesfollowed by centrifugation. Determination was made by isocratic ion chromatography using a DX500 system (Dionex) with IonPack CG12A and CS12A columns and20 mM methanesulfonic acid as eluent. The protein concentration in the extracts was quantified ua Bradford-type (1976) dye-binding microassay using a commercial Bio-Rad kit (Watford, UK) and bovine serumalbumin as standard.
Determination of amino acid profile
Amino acids were separated and analyzed by capillary electrophoresis using a Beckman-Coulter PA-800 system with laser-induced fluorescence detection (argon ion: 488 nm; Takizawa and Nakamura, 1998; Arlt et al., 2001). Extraction was carried out in an aqueous solution containing 1 M HCl and the supernatant obtained after centrifugation used for analysis. Samples were derivatized with fluorescein isothiocyanate and the separation was performed in a 50 μm i.d. x 43/53.2 cm fused-silica capillary at a voltage of 30 kV and a temperature of 20 ºC. The migration buffer was 80 mM borax (pH 9.2) containing45 mM α-cyclodextrin. Sample injection was accomplished by a pressurized method (5 s).
Isotopic analysis and C-N determination
δ15N, % N and % C were determined for shoot and root samples (approx. 1 mg dry wt) by isotope ratio mass spectrometry under continuous flow conditions. Samples were weighed, sealed into tin capsules (5 × 8 mm, Lüdi AG) and loaded into the autosampler of an NC elemental analyser NC 2500 (CE instruments, Milan, Italy). The capsule was dropped into the combustion tube (containing Cr2O3 and Co3O4Ag) at 1020 °C with a pulse of oxygen. The resulting oxidation products (CO2, NxOy and H2O) were swept into the reduction tube (Cu wire at 650 °C), where oxides of N were reduced to N2 and excess oxygen was removed. A magnesium perchlorate trap removed the water. N2 and CO2 were separated on a GC column (Fused Silica, 0.32 mm × 0.45 mm × 27.5 m, Chrompak) at 32 °C and subsequently introduced into the mass spectrometer (TermoQuest Finnigan model Delta plus, Bremen, Germany) via a Finnigan Mat Conflo II. δ (‰) Values were calculated as follows:
where R is the 15N/14N ratio.
The results were mathematically transformed and presented in terms of % 15N.
Statistical Analysis
All data collected were analysed statistically. Means were tested by applying Student's t test (p≤0.05; SPSS software, version 15), and significant differences between treatments (urea-fed plants vs. urea+NBPT-fed plants) are indicated by asterisks.
Results
No significant differences in dry weight were found with respect to control plants after 9 days' treatment with NBPT (Table 1), although pea plants showed some morphological changes. Thus, the growth of root at the expense of shoot was 50% higher in the case of NBPT-treated pea plants. Furthermore, the leaves on the lowest part of plants treated with the inhibitor showed leaf-tip scorch and necrosis. Indeed, the urease inhibitor caused a differential distribution of photosynthates, which translated into a significantly higher C/N ratio. In contrast, growth of spinach plants was not significantly affected by the application of NBPT, with no signs of scorch or necrosis and no changes in the C/N ratio.
the NBPT molecule was not detected in the tissues of control plants, whereas pea plants presentedhigher NBPT levelsthan spinach plants (35% higher) in both root and leaf upon treatment with urea + NBPT (Table 2).
The urease activity also differed between pea and spinach plants. Thus, although both species exhibited higher control values in roots than in shoots, pea plants presented fivefold higher values than spinach plants (Fig. 1). NBPT led to a dramatic reduction in urease activity in pea plant leaves,although the activity returned to control levels 7-9 days after treatment. In contrast to leaves, the effect of the inhibitor could be seen in pea plant roots throughout the entire treatment period, with no significant recovery by the end of the experiment. The effects of NBPT on urease activity in spinach plant leaves were not significant, and very small effects were seen in the roots. Indeed, and somewhat unexpectedly, NBPT treatment increased urease activity with respect to the control plants at some time points. Replacement of the solution at the onset of treatment in control pea plants led to an increased urease activity in leaves, although this returned to normal around day 8. This increase was not as significant in roots.
Internal urea levels were 10 times higher in control pea plants than in control spinach plants in both leaf and root (Fig. 2), although it should be noted that the concentration of urea in the growth solution for both species was different (5 mM urea for pea and 1.5 mM for spinach). Addition of NBPT to the growth solution led to an increase in urea levels, especially in leaf. This increase was particularly notable in pea plants, where urea levels in mature leaves were found to be 50 times higher than in control plants (30 times higher in young leaves; data not shown). The urea content in spinach plants also increased upon treatment with NBPT, although this increase was not as pronounced as that seen for pea plants.
One expected consequence of urease inhibition would be a reduction in ammonium levels due to a reduction in the hydrolysis of urea. This reduction was seen in the roots of pea plants,whereas no such effect was observed in spinach plants. In contrast,leaf ammonium content was higher in spinach plants treated with inhibitor than in control spinach plants(Fig. 3). Generally speaking, pea plants had higher ammonium levels than spinach plants (10 times higher in leaf and 20–30 times higher in root). The significant reduction of ammonium levels in pea plant roots was related to the significant drop in both amino-acid and soluble-protein levels (Fig. 4 and 5). A similar effect was observed in that part of the plant above the ground. Application of NBPT to spinach plants also resulted in a decrease in amino-acid and soluble-protein levels, although this decrease was much lower than that observed in pea plants.
Amide forms (i.e. glutamine and asparagine and their derivatives) represented more than 50% of the total amino acid content in the leaves of control pea plants, whereas this value in root was higher than 90% (Fig. 6). The greater reduction in the level of these amino acids upon treatment with NBPT is the main reason for the reduction of the total amino acid pool. This can readily be seen by considering asparagine, which went from being the most abundant amino acid in control plants to being undetectable in the leaves of plants treated with the inhibitor. Amide forms represented around 50% of the total amino acid content in the leaves of control spinach plants but only 25% in root. The decrease in these amino acids upon application of NBPT was only significant in the case of glutamic acid in root, which is the main amino acid in both spinach root and leaf. Despite the drastic reduction in the content of most amino acids, the levels of some of them, especially isoleucine and tryptophan, increased in pea roots and leaves.
NBPT reduced the incorporation of labelled urea in both plant species, as shown by the %15N values for roots, whilst the behaviour in leaf was different. Thus, whereas NBPT had no effect on %15N levels in pea plant leaves, higher %15N levels were found in control spinach plants than in those treated with inhibitor (Fig. 7).
Discussion
A tendency for reduction in growth of treated plants with respect to control plants was observed for both species nine days after the application of NBPT, although this reduction was not statistically significant. Biomass partitioning was also altered, with root/shoot ratio being notably higher in pea. The high impact of NBPT on the C/N ratio of pea plants suggests aninterference of NBPT with N availability in pea plants.
NBPT treatment drastically reduced shoot and root urease activity in pea plants, although this inhibition seems to be transient sinceurease activity in shoots returned to levels prior to NBPT application after seven days.This is in accordance with the results reported by Krogmeier et al. (1989), who found that urease activity was unaltered in wheat and sorghum leaves 21 days post-treatment. In contrast, the inhibition of root urease was maintained throughout this study. The effect of NBPT on urease activity in spinach was not significant, although the significantly different urea content indicates that NBPT has some effect. This different behaviour of the inhibitor as regards urease inhibition in these two species could be related to either its differential absorption in the two species and/or to structural differences between the ureases found in pea and spinach plants. Unfortunately, the three-dimensional structure of a plant urease has not yet been determined. However, various authors have reported a lower urease activity for canatoxin, a jackbean isoform, which could be related to the presence of one Zn atom per monomer at the enzyme's active site rather than two nickel atoms (Follmer et al., 2002; 2004). Canatoxin displays insecticidal activity against Coleoptera (beetles) and Hemiptera (bugs) (Carlini and Grosside-Sa, 2002). It is possible that the role of urease in spinach is mainly defensive, whereas in pea plants, due to their higher ureolytic and nitrogen-fixation ability, urease could allow the plant to use either externally or internally generated urea as a nitrogen source. The broad distribution of ureases in leguminous seeds, as well as the accumulation pattern of the protein during seed maturation, suggests an important physiological role for this enzyme (Follmer, 2008). The principal urea-generating route in plants is the arginase reaction, in which arginine metabolised into urea and ornithine. Arginine is an important constituent of proteins and an important N transport and storage compound in deciduous trees, conifers and seeds (Polacco and Holland, 1993). Urea can also be generated from ureide (allantoate, allantoin) catabolism. Indeed, it has been demonstrated that ureidoglycolate, a product of allantoate degradation, is a urea precursor (Todd and Polacco, 2004; Muñoz et al., 2006).