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Short running title: NBPT implications on nitrogen metabolism
Corresponding author: Pedro M. Aparicio-Tejo
Mailing address: Institute of Agri-biotechnology Institute (IdAB). UPNaCSIC-GN. 31192 Mutilva Baja. Navarra. Spain.
Phone: (+34)948168000.
Fax: (+34)948168930
E-mail: [email protected]
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. Public University 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. Public University 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 sole N 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 NBPT content 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 pea
being 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 activity in
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 allowing these
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, which delay the
hydrolysis process and thereby extend urea availability by avoiding nitrate
leaching and reducing 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 shortterm 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-free
solution) 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 their early 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
8 L/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 minutes followed by centrifugation. Determination was made by
isocratic ion chromatography using a DX500 system (Dionex) with IonPack
CG12A and CS12A columns and 20 mM methanesulfonic acid as eluent. The
protein concentration in the extracts was quantified u a Bradford-type (1976)
dye-binding microassay using a commercial Bio-Rad kit (Watford, UK) and
bovine serum albumin 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) containing 45 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. N 2
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:

Rsample  Rstd
1000
Rstd
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 presented higher NBPT levels than 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 an interference 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 since urease 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 posttreatment. 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 nitrogenfixation 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).
The peak in urease activity observed at days 1-2 in control pea plants could
be a consequence of the higher urea concentration due to renewal of the
nutrient solution at the beginning of the experiment. A similar induction of
urease activity has also been reported in barley leaves (Chen and Ching, 1988).
Although regulation of urease expression is not well understood in plants,
different routes for the regulation of this enzyme have been described in
bacteria, including regulation by the global N control system, induction by the
presence of the substrate urea, developmental regulation in Proteus species
and, finally, the urease in Streptococcus salivarus is reported to be regulated by
pH (Mobley et al., 1995).
As a consequence of the lower urease activity and the fact that a ureabased nutrient solution was used, NBPT-treated pea plants accumulated
considerable amounts of urea in their leaves. A similar effect has previously
been described by Krogmeier (1989), and some authors have suggested the
toxicity of urea to be the cause of leaf-tip scorch and necrosis (Gerendás and
Sattelmacher, 1999). In this study, the urea content in the leaves of pea plants
treated with inhibitor was 100-fold higher than that observed in control plants.
However, despite this significant accumulation, it is not possible to state
conclusively that this is the cause of the effects observed. Indeed, the almost
30-fold higher accumulation of urea observed in younger leaves (data not
shown) does not affect the aspect of these leaves with respect to those of the
control treatment. Watson and Miller (1996) have proposed that pH variations
resulting from the hydrolysis of urea upon recovery of urease activity could also
lead to the phytotoxicity observed in shoots. In our study, shoot urease activity
took seven days to recover. However, shoot urease activity in pea plants is
unlikely to be sufficiently important to produce the observed effect since the
ammonium levels resulting from urease activity at day 9 were not high enough
to support this hypothesis.
Although inhibition of urease activity in spinach upon application of NBPT
was not significant, a significant increase in urea concentration was observed,
although to a level below that found in pea plants, including untreated plants.
Surprisingly, urea levels were higher in leaves than in roots in the two
species studied, thus indicating that urea, which is a very small and soluble
molecule, can undergo fast translocation from the plant root to the shoot in the
transpiratory flow. The ammonium content in control pea roots increased in
response to the supplement of urea, whereas it decreased 10-fold in the NBPTtreated plants. This reduction extended from day 1 to the conclusion of the
study. In contrast, NBPT treatment resulted in higher ammonium levels in
spinach shoots, although these levels were always below those found in pea.
The part of the plant which shows altered ammonium content appears to
coincide with the site where ammonium is assimilated. Thus, the roots are the
main N-assimilatory organ in pea, whereas N assimilation takes place in shoots
in spinach (Lasa et al., 2002). Pea roots show greater levels of ureic hydrolysis
activity
and
subsequent
incorporation
of
the
ammonium
released.
Consequently, NBPT appears to act first on the root, which results in a drastic
reduction of urease activity, a reduction of ammonium content and therefore
inhibition of the N-metabolism. However, the effects of this N deficiency are first
noted in shoots, probably due to the accumulation of urea. In spinach plants, it
is possible that NBPT affects other routes. The NBPT could stimulate the
deamination of N compounds and the photorespiration or could inhibit the
routes of ammonium assimilation, which would explain the ammonium increase
observed after treatment with NBPT.
Urease inhibition in pea caused a drastic reduction in N metabolism, as
reflected in a decrease in the amino acid pool. By the end of the study this
decrease in amino acid content ranged from fivefold in leaf up to almost 20-fold
in the case of roots. A similar reduction was observed for protein content,
although this occurred more gradually. The fact that total protein content
decreases gradually during NBPT treatment may indicate an important
decrease in the de novo synthesis of amino acids. The reduction in N
metabolism in spinach as a result of NBPT treatment is reflected in the
decrease in total protein content, although this decrease is lower than in pea.
The principal amide in pea plants is asparagine, whereas in spinach plants it
is glutamine. This fact can be related to the site of ammonium assimilation,
since the ability of legume roots to export asparagine reflects an ability to
assimilate nitrogen that is not seen in the roots of other species (Oaks, 1992).
Our results show that these amino acids are the most affected by NBPT
treatment, with the reduction in the content of these amino acids upon NBPT
treatment being the main factor underlying the reduction of the total amino acid
pool in both cases.
The high asparagine content in control pea plants suggests a high activity
for asparagine synthetase, which catalyzes the transfer of the amido group from
glutamine to aspartate to generate glutamine and asparagine. Ammonium can
also act as a substrate for asparagine synthetase, although in a less effective
manner (Coruzzi and Last, 2000).
Ammonium from urea becomes available to the plant upon hydrolysis by
urease, therefore urea fertilization can be considered to be analogous to
ammoniacal fertilization, where asparagine synthetase plays an important role
in preventing ammonium from reaching toxic levels. The C/N ratio is one of the
factors known to regulate asparagine synthetase levels (Herrera-Rodriguez et
al., 2007), therefore a higher availability of N would stimulate its expression. In
this study, the addition of NBPT together with urea would limit the availability of
N and thereby inhibit asparagine synthetase, which may well explain the low
levels of asparagine observed.
Amino acids have a wide range of functions in plants and are also the
structural units from which proteins are made. Any disruption to N metabolism
that implies a variation in amino acid content is therefore likely to affect plant
growth and development.
Despite the drastic reduction in the content of most amino acids observed in
pea plants, some of them were found to be present at higher levels, as was the
case for isoleucine and tryptophan in roots or leaves. Tryptophan is a precursor
in the synthesis of indole-3-acetic acid (IAA), a phytohormone involved in
physiological processes such as apical dominance or the rooting of plant
cuttings, amongst others (Bandurski et al., 1993). The increased tryptophan
levels found in this assay upon application of NBPT could result in changes to
the level of auxins, which would help to explain the changes observed in the
root/shoot ratio of pea plants.
In conclusion, the urease activity inhibitor NBPT is applied with the aim of
decreasing soil microbial urease activity. Nevertheless, our study reveals that
NBPT is absorbed by the plants and produces changes in their nitrogen
metabolism. Moreover, these changes of nitrogen metabolism seem to be
dependent on the plant species under study.
Acknowledgments
This work was supported by the Spanish MICIIN (grant no. AGL2009-13339CO2-02 [to P.A.T.]). S.C was supported by a doctoral fellowship from the Public
University of Navarre.
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1979;31:189-223.
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Table 1. Dry weight (g), Root/Shoot and C/N ratios of pea and spinach plants at days 0
and 9. Data represent mean ± standard error (n=3). Asterisks represent significant
differences between treatments at day 9 (significance level of 95%).
Pea
Day 0
Dry weight
Root/Shoot
Control
Control
0.790 ± 0.05
+ NBPT
Control
C/N
Day 9
8.43 ± 0.33
+ NBPT
Day 0
1.276 ± 0.09
0.507 ± 0.09
+ NBPT
Spinach
1.189 ± 0.17
0.880 ± 0.10
1.253 ± 0.06*
6.98 ± 0.40
10.47 ± 0.62*
Day 9
1.073 ± 0.10
0.801 ± 0.07
17.33 ± 0.91
1.756 ± 0.02
1.394 ± 0.20
0.539 ± 0.00
0.558 ± 0.01
32.04 ± 2.77
38.12 ± 4.69
Table 2. NBPT content (μmol g-1 DW) in leaf and root of pea and spinach plants at day
2, after the application of the treatments, n.d. = not detected. Data represent mean ±
standard error (n=2).
Pea
Spinach
Leaf
Root
Leaf
Root
Control
n.d.
n.d.
n.d.
n.d.
+ NBPT
2.565 ± 0.14
0.063 ± 0.01
1.647 ± 0.17
0.041 ± 0.00
PEA
SPINACH
150
150
(U g-1 prot)
50
*
(U g-1 prot)
*
*
root
0
150
root
100
100
*
50
*
*
1
2
*
*
*
*
Urease activity
50
Urease activity
100
(U g-1 prot)
100
0
150
Urease activity
leaf
(U g-1 prot)
Urease activity
leaf
0
50
0
0
3
4
5
6
7
8
9
0
1
2
3
Days
4
5
6
7
8
9
Days
Fig. 1. Urease activity in leaf and root of pea and spinach plants at days 0, 1, 2, 4, 7
and 9 after the application of the treatments: urea-fed plants (○), urea + NBPT-fed
plants (●). Data represent mean ± standard error (n=6). Asterisks represent significant
differences between treatments (significance level of 95%).
PEA
leaf
50
30
*
200
20
100
*
*
*
*
*
*
*
*
0
(mol g-1 DW)
10
0
75
Urea content
(mol g-1 DW)
300
Urea content
40
(mol g-1 DW)
400
Urea content
(mol g-1 DW)
*
leaf
500
Urea content
SPINACH
root
*
60
7,5
root
*
6,0
*
*
45
*
*
30
4,5
3,0
*
15
1,5
0
0,0
0
1
2
3
4
5
Days
6
7
8
9
0
1
2
3
4
5
6
7
8
9
Days
Fig. 2. Urea content in leaf and root of pea and spinach plants at days 0, 1, 2, 4, 7 and
9 after the application of the treatments: urea-fed plants (○), urea + NBPT-fed plants
(●). Data represent mean ± standard error (n=9). Asterisks represent significant
differences between treatments (significance level of 95%).
*
15
10
1,5
1,0
*
*
5
0,0
root
240
(mol g-1 DW)
Ammonium content
0
0,5
(mol g-1 DW)
2,0
leaf
*
Ammonium content
leaf
(mol g-1 DW)
(mol g-1 DW)
SPINACH
Ammonium content
Ammonium content
PEA
20
root
24
180
18
120
12
60
*
*
1
2
6
*
*
*
*
0
0
3
4
5
6
7
8
0
9
0
1
2
3
4
5
6
7
8
9
Days
Days
Fig. 3. Ammonium content in leaf and root of pea and spinach plants at days 0, 1, 2, 4,
7 and 9 after the application of the treatments: urea-fed plants (○), urea + NBPT-fed
plants (●). Data represent mean ± standard error (n=3). Asterisks represent significant
differences between treatments (significance level of 95%).
PEA
0,4
0,4
0,3
0,3
0,2
0,2
*
0,1
*
0,1
(mmol g DW)
-1
Amino acids content
0,0
1,5
(mmol g-1 DW)
(mmol g DW)
0,5
leaf
Amino acids content
leaf
(mmol g-1 DW)
-1
SPINACH
Amino acids content
Amino acids content
0,5
0,0
root
0,15
root
1,2
0,12
0,9
0,09
0,6
0,06
*
0,3
*
*
*
*
0,03
*
*
0,0
0
1
2
3
4
5
Days
6
7
8
9
0
1
2
3
4
5
6
7
0,00
8
9
Days
Fig. 4. Amino acids content in leaf and root of pea and spinach plants at days 0, 1, 2,
4, 7 and 9 after the application of the treatments: urea-fed plants (○), urea + NBPT-fed
plants (●). Data represent mean ± standard error (n=3). Asterisks represent significant
differences between treatments (significance level of 95%).
PEA
150
75
*
*
100
50
*
50
100
25
*
*
*
root
*
0
50
root
40
80
(mg g-1 DW)
(mg g-1 DW)
100
Protein content
200
0
Protein content
125
(mg g-1 DW)
(mg g-1 DW)
leaf
leaf
Protein content
Protein content
250
SPINACH
60
30
*
40
*
*
20
*
*
*
*
20
10
*
0
0
0
1
2
3
4
5
Days
6
7
8
9
0
1
2
3
4
5
6
7
8
9
Days
Fig. 5. Protein content in leaf and root of pea and spinach plants at days 0, 1, 2, 4, 7
and 9 after the application of the treatments: urea-fed plants (○), urea + NBPT-fed
plants (●). Data represent mean ± standard error (n=9). Asterisks represent significant
differences between treatments (significance level of 95%).
PEA
3
6
2
4
*
1
2
Asn
Gln
0
Glu
0
Asp
0
8
Ala
*
*
Gln
Ala
Gaba
Gly-Ser
Thr
Val
Pro
Tyr
His
Trp
Met
Phe
Ile
Leu
Lys
Arg
0
*
Asn
100
*
4
Gaba
*
*
*
Glu
1
root
Gly-Ser
200
*
0
10
Thr
*
*
Pro
*
2
0
5
Tyr
300
*
6
Val
3
3
His
400
Asp
(mol g-1 DW)
4
12
Trp
root
6
Phe
0
500
18
Ile
60
*
*
9
Met
*
24
Leu
*
*
(mol g-1 DW)
*
Amino acids profile
*
3
12
Lys
120
*
30
leaf
Arg
6
(mol g-1 DW)
180
Amino acids profile
240
9
0
5
Amino acids profile
leaf
12
(mol g-1 DW)
Amino acids profile
25
SPINACH
15
300
Fig. 6. Amino acids profile in leaf and root of pea and spinach plants at the end of the assay. Control (□); + NBPT (■). Data represent mean ±
standard error (n= 3). Asterisks represent significant differences between treatments (significance level of 95%).
PEA
4
3
3
15N
2
*
*
root
*
*
*
*
1
0
5
root
3
3
2
2
*
*
*
*
*
*
*
1
2
*
*
*
0
(%)
4
content
4
15N
(%)
2
*
*
1
(%)
4
0
5
content
5
leaf
content
(%)
leaf
1
15N
SPINACH
15N
content
5
1
0
0
1
2
3
4
5
Days
6
7
8
9
0
3
4
5
6
7
8
9
Days
Fig. 7. Percentage of 15N in leaf and root of pea and spinach plants at days 0, 1, 2, 4, 7
and 9 after the application of the treatments: urea-fed plants (○), urea + NBPT-fed
plants (●). Data represent mean ± standard error (n= 3). Asterisks represent significant
differences between treatments (significance level of 95%).