Short running title: NBPT implications on nitrogen

metabolism

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: pmapariciotejo@unavarra.es

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 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-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. 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 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 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).

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 urea-based 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 NBPT-treated 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.

AGL2009-13339-CO2-02 [to P.A.T.]). S.C was supported by a

doctoral fellowship from the Public University of Navarre.

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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 SpinachDay 0 Day 9 Day 0 Day 9

Dryweight

Control0.507 ±

0.09

1.276 ±0.09 1.073 ±

0.10

1.756 ±0.02

+ NBPT 1.189 ±0.17

1.394 ±0.20

Root/Shoot

Control0.790 ±

0.05

0.880 ±0.10 0.801 ±

0.07

0.539 ±0.00

+ NBPT 1.253 ±0.06*

0.558 ±0.01

C/NControl

8.43 ± 0.33

6.98 ± 0.4017.33 ±

0.91

32.04 ±2.77

+ NBPT 10.47 ±0.62*

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 SpinachLeaf 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

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%).

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%).

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%).

Amino acids content

(mmol g

-1 DW)

0,0

0,1

0,2

0,3

0,4

0,5

Days0 1 2 3 4 5 6 7 8 9

Amino acids content

(mmol g

-1 DW)

0,0

0,3

0,6

0,9

1,2

1,5

**

**

* *

PEAAm

ino acids content(mmol g

-1 DW)

0,0

0,1

0,2

0,3

0,4

0,5

Days0 1 2 3 4 5 6 7 8 9

Amino acids content(mmol g

-1 DW)

0,00

0,03

0,06

0,09

0,12

0,15

SPINACH

* **

leaf leaf

root root

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%).

Prot

ein

cont

ent

(mg

g-1

DW)

0

50

100

150

200

250

**

*

Days

0 1 2 3 4 5 6 7 8 9

Prot

ein

cont

ent

(mg

g-1

DW)

0

20

40

60

80

100

**

*

**

Protein content(m

g g -1 DW)

0

25

50

75

100

125

Days

0 1 2 3 4 5 6 7 8 9

Protein content(m

g g -1 DW)

0

10

20

30

40

50

*

*

*

*

** *

leaf leaf

root root

SPINACHPEA

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%).

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 differencesbetween treatments (significance level of 95%).

15N content

(%)

0

1

2

3

4

5

Days

0 1 2 3 4 5 6 7 8 9

15N content

(%)

0

1

2

3

4

5

PEA SPINACH15N content

(%)

0

1

2

3

4

5

Days

0 1 2 3 4 5 6 7 8 9

15N content(%

)

0

1

2

3

4

5

leaf leaf

root root

* * * * *

* * * * *

** *

* * * * *

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%).