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The Signal Role of Nitrate in the Correction and Improvement of the Negative
Effects of Ammonium and /or Urea Nutrition on Plant Development

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Chapter 5

THE SIGNAL ROLE OF NITRATE

IN THE CORRECTION AND IMPROVEMENT
OF THE NEGATIVE EFFECTS
OF AMMONIUM AND / OR UREA NUTRITION
ON PLANT GROWTH AND DEVELOPMENT

M. Garnica1, F. Houdusse1, S. San Francisco1,

A. M. Zamarreño1, O. Urrutia1, R. Baigorri1,
J. C. Yvin1 and J. M. García-Mina1,2*
R&D CIPAV – Timac Agro Roullier Group; Poligono Arazuri-Orcoyen,
1

C/C, Orcoyen, Spain

2
Department of Chemistry and Soil Chemistry (Agricultural Chemistry),
Faculty of Sciences, University of Navarra, Pamplona, Spain

ABSTRACT
The aims of this chapter are: (i) First, to present new experimental evidence showing
the signal-role of nitrate in the correction and improvement of the deleterious effects of
ammonium- and/or urea-based nutrition in different plant species, either dicotyledonous
or monocotyledonous. Likewise, we discuss the different and complementary
mechanisms, which are expressed at diverse but interconnected molecular and
physiological levels, responsible for this specific role of nitrate. This discussion is
accompanied by the proposition of different hypothesis that could explain these
experimental results and orientate future research in this important field as well. (ii)
Second, we also discuss the different methodologies that might be developed in order to
apply this basic research in the development of new nitrogen fertilizers with higher
metabolic efficiency and lower potential environmental risks. (iii) Finally, we propose a
framework for future research oriented to do a synthesis -involving and inter-connecting

*
E-mail: mgarnica@timacagro.es; jgmina@timacagro.es.
68 M. Garnica, F. Houdusse, S. San Francisco et al.

this basic and applied research - related to both the future development of nitrogen-
related fertilizers and crop yield – quality improvement.

INTRODUCTION
Several studies have shown that nitrogen (N) is the mineral nutrient that has more impact
on crop yield and quality [1, 2]. This fact seems to be associated not only with the special role
of this nutrient in the regulation of plant hormone metabolism [3], but, also, because
fertilizers based on different N-sources suffer many losses in the field, which directly affect
their nutritional efficiency [4]. In addition, as it will be discussed below, these losses have
negative consequences on the environment, either by affecting climate global change (the
release to the atmosphere of N-oxides) [4-6], or by increasing the contamination of natural
water reservoirs [7].
The main sources of N used to produce mineral N-based fertilizers are nitric-N (NT)
(nitric acid and nitrate salts); ammonium-N (A) (ammonium salts: sulphate, phosphate,
nitrate…) and urea-N (U) [8]. Some studies have also reported that some organic forms of N
(mainly, some types of amino acids) may be alternative nutritional sources for plants [9, 10].
However the practical value of these organic forms is, so far, very low.
However, the main three above-mentioned N-forms have some limitations that affect
their nutritional value as N source for plants. These limitations are related to both plant
physiological issues (mainly, root uptake and further assimilation within the plant) [11] and
losses in field (volatilization, leaching, soil fixation-absorption …) [4, 6]. However, only
those limitations related to N losses and potential contamination of natural ecosystems have
received special attention for farmers and governments [6, 12].
The losses associated with the field application of N-based fertilizers are, mainly, NT
leaching and denitrification [4, 7]; ammonia - volatilization (principally from the urea
hydrolysis in the soil) [4, 13], and U-leaching [7] (Figure 1).

Figure 1. Nitrogen cycle in soils.

 The Signal Role of Nitrate in the Correction and Improvement … 69

It becomes clear that all these processes are strongly affected by several factors such as
soil texture and physico-chemical properties (mainly pH, clay content, organic matter-
microbial activity), climate (rainfall and temperature), the type of fertilizer (fertilizer features:
granule or powder; coated …), the type of fertilizer application to the field, or the type of crop
management (tillage, non-tillage …) [13].
In this context, in order to decrease the impact of these potential losses on N-fertilizer
efficiency and the environmental contamination, a number of techniques have been developed
[7]. Among these techniques, the most relevant are: the use of water-insoluble organic N
compounds (urea-based polymers) (UP) [7]; the use of slow-release coated N fertilizers
(SRC) [7]; and the use of inhibitors of soil urea hydrolysis (UIH) or soil ammonium
nitrification (AIH) [7, 13] or both in the same N fertilizer (Table 1).

Table 1. Types of special N fertilizers

Slow-release nitrogen
fertilizers
Coated materials Uncoated inorganic Uncoated organic materials
materials
(Soluble fertilizers) (Insoluble (Insoluble fertilizers)
fertilizers)
Sulfur-coated urea (SCU) NPK stake Ureaforms (UF)
Sufur-coated NPK fertilizers NPK spike Crotonylidine diurea (CDU)
Polymer-coated urea Magnesium Isobutylidene diurea (IBDU)
ammonium
phosphate
Polymer-coated NPK Potassium Melamine (triaminotriazine)
fertilizers ammonium
phosphate
Petroleum-coated urea Melamine + urea
Petroleum-coated NPK Ca Oxamine
Polyethylene NPK packets Guanylurea phosphate Guanylurea sulfate
Urea based fertilizers with urease inhibitor
NBPT
Ammonium based fertilizers with
nitrification inhibitor DCD
Ammonium based fertilizers with
nitrification inhibitor DMPP

However, in the case of industrial, intensive or extensive, crops (such as cereals,

leguminous …) the use of UP- or SRC- based N fertilizers is not suitable because the rate of
release of plant available N from the fertilizer does not match N crop demands [8]. In these
cases, the use of U-fertilizers amended with UIH or A-fertilizers amended with AIH has
proven to be partially efficient [7, 13].
However, the use of these inhibitors not only has environmental consequences but also
raises new questions regarding plant physiology and mineral nutrition. In effect, whereas in
agricultural soils the main N form available for plants is NT [2, 13], when UIH or AIH are
used, U, A, and mixed forms containing U, A and NT become alternative N forms for plants
[2, 7, 13]. In this context those questions related to the real efficiency of these new N-forms,
70 M. Garnica, F. Houdusse, S. San Francisco et al.

different from NT alone, as N nutritional sources for plants have great interest. Furthermore,
the knowledge obtained from these studies may be very relevant in order to reduce the
amount of NT to be used in crop fertilization, by optimizing the nutritional value of A and/or
U. This reduction of NT has – potentially - many advantages, mainly concerning
environmental issues such as global climate change (the emission of N-oxides to the
atmosphere) [6], and the contamination of natural water reservoirs with nitrate [7].
In this framework, the aim of this chapter is to discuss the nutritional efficiency in plants
of N-sources different from NT, such as A, U and mixed N-forms containing NT, A and/or U
simultaneously; and the potential practical application of these findings for the development
of new N-based fertilizers.

EFFICIENCY OF NITRATE, AMMONIUM AND UREA

AS N-NUTRITIONAL SOURCES FOR PLANTS
Regarding the plant uptake and further assimilation within the plant of the above-
mentioned three main N-sources (NT, A and U), numerous studies reported that the efficiency
of NT is clearly higher than that of A or U, when these N-forms are supplied separately
[14-17]. The main mechanisms involved in NT root uptake and further assimilation within the
plant are presented in Figure 2.
These mechanisms contemplate two different scenarios: (i) in case of low availability of
NT (<1 mM) in the soil or nutrient solution, the plant activates a specific mechanism named
HATS (high affinity transport systems), which involves the induced-production of several
NT-transporters in the root [11] coded by the NRT2 genes family [18, 19]; (ii) in case of high
NT (>1 mM) availability in the soil or nutrient solution, plant activates another mechanism
named LATS (low affinity transport systems) , which involves several NT-transporters in the
root, different from those activated in HATS [11].
These low affinity transporters are coded by the NRT1 genes family [19, 20]. Likewise,
the translocation of NT from the root to the shoot has important physiological implications in
shoot growth, because NT trafficking inside the plant plays a role of functional signaling in
the enhancement of the root to shoot translocation of active forms of several cytokinin (CK)
types [3, 21-23], and other plant phytoregulators, such as indole-acetic acid (IAA) and
abscisic acid (ABA) [23] (Figure 3).
As for NT metabolism, this N form is converted into A by the enzymatic system named
Nitrate Reductase (NR), which, in turn, involves two enzymes: NR to reduce NT to nitrite,
and Nitrite Reductase (NiR) to reduce nitrite to A [11] (Figure 4).
The A produced from NT reduction is incorporated into amino acids by the enzymatic
system named GS (Glutamine synthetase)-GOGAT [11]. Many of these amino acids are
involved in the regulation of the biosynthesis of the main phytoregulators, - such as
polyamines, ethylene, cytokinins, and auxins – and other secondary metabolites with
physiological relevance [11] (Figure 3).
As for A root uptake and further assimilation within the plant, several studies have
characterized some of the transporters present in root responsible for the transport of A into
root cells [11]. Transport of A across membranes is mediated by proteins of the
AMT/MEP/Rh family [24]. In Arabidopsis, AMT2.1 is the only member of the MEP
 The Signal Role of Nitrate in the Correction and Improvement … 71

subfamily, while five homologs, AMT1.1 to AMT1.5, constitute the AMT clade [25]. Four of
the homologs are expressed in roots and upregulated under nitrogen deficiency [26-29]
(Figure 2). Ammonium is further incorporated into general metabolic pathways through the
formation of glutamine (Gln) by the enzymatic system GS-GOGAT, mainly in the root but
also in the shoot (Figure 4) [11].

Figure 2. Mechanisms of transport and uptake of NO3-(A), NH4+ (B) and urea (C) in Arabidopsis.

Figure 3. Different steps of nitrogen metabolism in plants. Biosynthetic pathways of amino acids and
phytoregulators where nitrate can exert an effect as a signal molecule.
72 M. Garnica, F. Houdusse, S. San Francisco et al.

Figure 4. Main enzymatic pathways involved in NO3-, NH4+ and urea assimilation. NR: Nitrate
Reductase, NiR: Nitrite Reductase, GS: Glutamine synthetase, GOGAT: Glutamate synthetase, AS:
Asparagine synthetase, GDH: glutamate dehydrogenase.

This process of transformation of A in Gln is crucial for the correct development of the
plant. This is because A-accumulation causes severe toxicity that results in lower plant
growth, membrane depolarization, cellular damage and even, plant death (Figure 5) [11, 30,
31]. The mechanisms behind A toxicity in plants are complex, seem to be inter-connected,
and are only partially known [30, 31]. They involve apoplast acidification and the production
of some metabolites mainly synthesized by the plant to cope the stress caused by A local
accumulation in plant tissues [30, 31]. Several studies showed that there is an important
correlation between the intensity of the toxicity caused by A and polyamines accumulation,
principally putrescine (Put) in both wheat and pepper plants [31, 32]. This fact was upgraded
when Put was accumulated in the shoot, mainly, in the leaves [31]. The deleterious effect of
Put accumulation has been described in various studies [31].Sung et al. [33] reported that Put
accumulation inhibited the growth of rice cells under potassium limiting conditions. Other
studies showed that Put accumulation in plant tissues is associated with negative effects on
plant development, such as depolarization of membranes, potassium leakage, protein loss, and
tissue necrosis [34]. In these sense, Minocha et al. [35] reported that overproducing transgenic
poplar cells presented greater plasma membrane damage and less tolerance to N-nutrition
containing A.
Other authors suggested that A toxicity in plants may also be associated with specific
changes in the hormonal balance within the plant, mainly linked to pH control in apoplast and
symplast [15, 31, 32]. In any case, more studies are needed to elucidate the intricate
mechanistic network involved in A-toxicity and A -toxicity control and regulation in plants.
As for U plant nutrition, studies are less numerous than in the case of NT or A. Some
experiments have shown that plants can assimilate U by the roots and leaves [17, 31, 36-40],
and that this molecule is adequately metabolized by plants through the activation of plant
ureases, when Ni deficiency does not occur [11, 40] (Figure 4). Likewise the existence of
specific transporters for urea in plant tissues has also been well established [11, 40]. Besides
plasma membrane localized aquaporins (PIP, NIP or TIP) the AtDUR3 represents the major
high affinity urea transporter in Arabidopsis [41] (Figure 2). However, as in the case of A-
 The Signal Role of Nitrate in the Correction and Improvement … 73

nutrition, some studies reported that U accumulation in plant tissues resulted in lower growth
and changes in leaf texture and colour [17, 31, 39, 42] (Figure 5).

Figure 5. Negative effects of NH4+ (A) and urea (B) nutrition in wheat plants compared to NO3-
nutrition.

THE SIGNAL ROLE OF NITRATE IN THE CORRECTION

OF THE NEGATIVE EFFECTS OF AMMONIUM
AND UREA ON PLANT DEVELOPMENT

A number of studies have shown that the association of NT along with A (NA N-
nutrition) led to a significant improvement of the nutritional action of whole N-nutrition in
several plant species [30, 43]. Other studies have also shown that the presence of U affected
the assimilation of the other forms of N as well as that of other mineral nutrients [17, 39]. In
this sense, recent studies reported that the presence of NT along with A and U (NAU mixed
N-nutrition) significantly improved the shoot and root dry matter production of wheat and
pepper [17]. Surprisingly, these studies showed that NAU nutrition presented a whole
efficiency (expressed as the production of shoot dry matter by 1 % of N concentration in the
shoot) even significantly higher than that of NT [17]. These findings indicated that a potential
way to correct the negative effects of A and U on plant growth is the association of these N-
forms with NT in a sole N-nutrient supply.
As for the mechanisms that could explain this positive action of NT on A and/or U N-
nutrition, several studies have shown that, at least, there exist four complementary pathways
[17, 23, 31, 32, 44]: (i) a nutritional effect [17]; (ii) a protective effect by reducing free Put
74 M. Garnica, F. Houdusse, S. San Francisco et al.

accumulation and ethylene production ([31, 32, 44]; (iii) a positive action on A- and U- root
uptake rates and further assimilation within the plant ([39, 42]; (iv) a growth-promoting
effect, mainly related to an enhancement of root to shoot translocation of active forms of
several CK types [23, 44].

(i) Nutritional Effect

The studies carried out by Houdusse et al [17] in both a dicotyledonous plant species
(pepper) and a graminaceous plant species (wheat) showed that those plants fed with NAU- or
NA- nutrition presented a concentration of phosphorus (P) and iron (Fe), in the shoot (wheat)
and leaves (pepper), significantly higher than that of NT. Likewise, the study of the total
extraction of both nutrients as well as their relative shoot: root ratio indicate that the presence
of NT enhanced the root to shoot translocation of P and Fe [17].
These facts can be explained by the acidifying action of A, derived from the proton
release to the apoplast associated with A cell uptake, in plant tissues and xylem-related
channels, which can neutralize the alkalinizing action derived from proton-NT co-transport
into cells [11].
This pH acidification could lead to the solubilisation of water-insoluble Fe-
oxides/hydroxides and inorganic salts (iron phosphates); and water-insoluble phosphates
(calcium phosphates, aluminium phosphates or iron phosphates) [17].
This beneficial effect of NAU- and NA- N-nutrition has potential practical application in
the development of products for the correction of both P and Fe deficiencies. In the case of
wheat, the beneficial action of NT was also reflected in an increase in the concentration in the
shoot of magnesium (Mg) and calcium (Ca) [17].

(ii) Protective Effect

Recent studies have found a significant correlation between A-toxicity (expressed in the
reduction of dry matter production) and the concentration of free Put in the aerial part of
several plant species [31]. These results suggested that the beneficial action of the association
of NT with A and U could be related to a reduction of Put accumulation in the plant, mainly
in the aerial part.
This decrease in free Put might be caused by a reduction in Put synthesis, the formation
of conjugated forms of Put, a transformation of Put into spermidine (Spd) and spermine
(Spm), and/or an increase in Put catabolism. Houdusse et al [31] showed that NAU-fed plants
(both pepper and wheat) presented a lower concentration of free Put in the shoot than A-fed
plants. Indeed, the concentration of free Put in the shoot of NAU fed plants was similar to that
of NT- fed plants. This fact suggested the presence of an effect of NAU nutrition on free Put
metabolism. Further studies proved this hypothesis.
Thus, Houdusse et al. [32] reported that the association of NT with A caused a decrease
in free Put accumulation in the shoot of both pepper and wheat by increasing the formation of
non-covalent conjugated Put forms and decreasing free Put biosynthesis. All these effects of
NT on A- and U- N nutrition were reflected in a significant decrease in the Put : (Spm + Spd)
 The Signal Role of Nitrate in the Correction and Improvement … 75

ratio, (a ratio that is directly related to stress intensity), with respect to A- and AU- fed plants
[31, 32].
This fact may be explained by both a decrease in Put biosynthesis and an increase in the
conversion of Put into Spd and Spm [31, 32]. In fact, in both plant species, the decrease in
free Put concentration in the aerial part was correlated to an increase in plant growth [31, 32].

(iii) Enhancement of A- and U- Root Uptake Rates and Further Assimilation

within the Plant

Other studies, recently published, showed that the presence of NT along with A (NA) or
U (NU) in the nutrient solution supplied to wheat seedlings increased very significantly the
uptake rate of A (x 1.72), or U (x 5.31). In the case of NAU nutrition, the presence of NT
caused an increase in the root uptake rates of both A (x 3.23) and U (x 2.83) [39]. These
results might be the consequence of a reduction in A or U potential toxicity by NT; thus,
inhibiting the expression of those mechanisms related to the repression of the uptake of A and
U. However, the mechanism responsible for the action of NT to increase A and U root uptake
is not clearly elucidated yet.
Complementary studies, showed that this increase in A and U root uptake rates, caused
by the presence of NT, was associated with a concomitant increase in both A and U
assimilation in the root and shoot of wheat seedlings [39, 42]. However, this fact was only
observed in plants fed with NAU and not in those plants fed with NA [39, 42]. The study of
the different enzymatic systems involved in the assimilation of NT (NR), A (Glutamine
synthetase, GS) and U (Urease) showed that the increase in A and U assimilation are
correlated with a prompt increase in the activity of GS and urease respectively, during the
first hour of treatment with NAU nutrient solution [42]. However, the involvement of
alternative pathways for A assimilation different from GS activity are also possible, such as
the Glutamate dehydrogenase (GDH) reaction [42, 45]. In any case, taken together, these
studies indicate that the presence of NT- in NAU-nutrition improved A and U root uptake and
further assimilation in root and shoot of wheat plants. This fact help us explain the whole
beneficial action of NT on the nutritional value of A and U, mainly in formulations containing
the three N forms simultaneously.

(iv) Growth-Promoting Effect Related to NT-Induced Changes in the Plant

Distribution of the Main Plant Hormones

Finally, some studies had proposed that the beneficial effect of NT on the nutritional
value of A and/or U could be related to changes in the hormonal balance within the plant [11,
15, 31]. In this sense, several studies have shown that NT can play a role as a hormone-like
signal, promoting shoot growth through the increase in the concentration of the main active
CK in the shoot [3, 22]. Another complementary study demonstrated that this action of NT
was mainly due to an increase in the root to shoot translocation of active CK types (Zeatine-
based CKs and adenosine-based CKs) [21]. On the other hand, other studies also have
reported effects of NT on IAA and ABA concentration in the shoot of various plant species
76 M. Garnica, F. Houdusse, S. San Francisco et al.

[11]. It is, therefore, possible that the beneficial action of NT on A- and U- root uptake-
assimilation and toxicity also involves changes in the plant distribution of CKs and other
phytoregulators. Studies carried out in our laboratory have validated this hypothesis [23, 44].
Thus, in order to explore the mechanisms of NT beneficial effect on the nutritional value of A
or U, we investigated in wheat seedlings the effects of NT, supplied along with A, on: (i) free
and conjugated polyamine concentrations in roots and shoots; (ii) ethylene biosynthesis in
roots; (iii) CKs concentration in roots and shoots; and (iv) IAA and ABA concentrations in
roots and shoots. To this end we applied A (5 mM) along with two different doses of NT (100
µM and 5 mM). These NT-doses were selected in order to determine whether the beneficial
action of NT on A-nutritional value requires significant doses (nutritional character), or can
be promoted by very low doses (hormone-like signal - character). The results obtained
showed that the presence of both doses of NT, along with A, caused a pattern of Put, Spd and
Spm plant distribution quite similar to that of NT when applied alone [44]. This effect was
expressed in a prompt increase in free Spd that was well correlated to a decrease in ethylene
root production [44]. This result was expected as Spd and ethylene share a common
biosynthetic pathway (Figure 3). In addition, this increase in free Spd was well correlated
with changes in the pool of conjugated PAs [44]. In roots, however, these changes might
involve alternative mechanisms [44]. The above-discussed effect of NT, decreasing free Put
concentration and increasing the conjugated forms of this PA in shoots [32], was only clearly
observed for the high concentration of NT (5 mM). This result was in line to those reported
by Houdusse et al. [32] since these authors also employed a high concentration of NT in their
experiments. In conclusion, the profile of PAs associated with the beneficial action of NT
improving the growth of A-fed plants was in line with that of NT-fed plants, thus
corroborating the involvement of these regulators in NT beneficial effect on plants grown
with A. Regarding the plant distribution of the main CKs, IAA and ABA, results obtained
showed that the application of the two doses of NT along with A, was associated with an
increase in the concentration in shoots and roots of the main active forms of CKs (zeatine (Z),
trans-zeatine riboside (tZR), isopentenyl adenosine (IPR)) and a reduction in the levels of
inactive CKs forms (cis-zeatine riboside (cZR)) [23]. A similar effect was observed in
relation to IAA-shoot concentration [23]. Thus, the presence of NT increased the
concentration of IAA in the shoot, although the pattern of this increase was different
depending on NT dose. The higher dose caused a slow and sustained increase while the
lowest one caused a prompt but transient increase [23]. In the case of ABA, the two doses of
NT tended to decrease the concentration in shoots and roots [23]. This fact may be the
consequence of a decrease in the stress in A –fed plants, associated with the presence of NT.
It was noteworthy that the action of the lowest dose of NT is mainly expressed by prompt
and transient effects. This fact is consistent with a potential role of low doses of NT as an
hormone-like signal [3, 21-23]. However, in the case of the highest NT dose these effects are
normally also expressed but slowly and time-sustained [23]. In any case, there exists a
qualitative similarity between the effects caused by the two NT doses [23].
Taken together, all these findings indicate that the signal role of NT, mainly expressed in
the increase in the concentration of both active-CKs and IAA in the shoot [3, 21, 22], is also
involved in the positive action of NT on the nutritional effects of A-based nutrition in plants
[23]. Nevertheless, together with this plant growth promoting action, other complementary
actions – nutritional and protective against the stress caused by strict A nutrition – are also
involved. All these, probably integrated, mechanisms are presented in Figure 6.
 The Signal Role of Nitrate in the Correction and Improvement … 77

In conclusion, these results show that a N-fertilizer based on A and/or U may be very
efficient provided with a small amount of NT either in the fertilizer, or in the soil in which
this fertilizer is applied. However, as it will be discussed below, the two above-mentioned
conditional situations are complicated to be achieved.
Thus, numerous studies have demonstrated that the manufacture of granulated fertilizers
containing NT, A and U simultaneously is very complicated and has inherent risks, mainly
related with the high oxidant character of NT [8].
As for the presence of NT in soil, in general most agricultural soils have a small but
significant amount of NT [7]. However, the application of N-based fertilizers containing A
and/or U normally causes a very high local concentration of A (and/or U) in soil that is
associated with both a local inhibition of A-nitrification and an imbalance in the ratio NT:A
(and/or U) [7]. These two facts affect negatively the expression of the above-discussed
positive role of NT improving the nutritional value of A and/or U as N sources for plants.

Figure 6. Mechanisms involved in the positive action of NO 3- on the nutritional effects of NH4+ and
urea nutrition in plants.
78 M. Garnica, F. Houdusse, S. San Francisco et al.

FUTURE RESEARCH
In this context, one important question that has to be addressed in future research studies
is: How can we assure the presence of a fraction of NT that is able to correct and improve the
efficiency of A and U as N-forms for plants?
Concerning this question, in general the main problem that we have is that either A or U
are rapidly transformed in NT in soils [7]. Although this fact depends on soil features,
moisture and temperature; as these fertilizers are normally applied at sowing there are always
time enough to have a significant A- soil nitrification when plants begin to take up nutrients
from soil solution [7]. In principle, there are two technologies that can, at least potentially,
help us control the processes of U-hydrolysis and A-nitrification in soils: (i) the use of coated
U and/or A-based N fertilizers; and (ii) the use of urease and/or nitrification inhibitors.
The use of coated N fertilizers containing U and/or A (for instance, as ammonium
sulphate, ammonium phosphate or ammonium nitrate) may help control the availability of U
and A to soil ureases and nitrifying bacteria respectively [7]. This fact might inhibit soil U
hydrolysis rate and soil A nitrification rate, thus permitting the gradual production of NT in
soil. However, a number of studies have shown that these types of fertilizers, coated with
either synthetic polymers or mineral water-insoluble compounds (sulphur; calcium
sulphate…), are not adequate to assure the correct supply of N to annual-extensive (or
intensive) crops [7]. The fact is that this type of coating is not sensitive to plant nutritional
needs expressed through the root, but to environmental factors such as temperature and soil
moisture [7].
On the other hand, the application of urease inhibitors and/or A-nitrification inhibitors in
fertilizers is quite common [4]. However, the main problem associated with their use is that
the recommended doses per ton to be used in fertilizer-manufacture have been calculated in
order to solve (or minimize) environmental problems - ammonia volatilization in the case of
urease inhibitors, or nitrate leaching and denitrification in the case of A nitrification inhibitors
– without explicitly considering the physiological efficiency in plants of those fertilizer
amended with these compounds.
Thus, the use of soil urease inhibitors has been principally developed in countries with
environmental conditions that favour ammonia volatilization [13, 46]. There exist a number
of molecules that have shown satisfactory activity as urease inhibitors in soils (Table 1).
Among these molecules, phosphor-(n)-amidates derivatives have proven to have high ability
to inhibit ammonia volatilization, mainly in alkaline soils [13, 46-48]. A number of studies
have reported that the efficiency of urease inhibitors depends on many external variables,
such as soil physico-chemical properties (mainly pH, clay content and organic matter-
microbial activity), temperature and soil water content [13, 46]. These facts make it very
difficult to calculate the concentration of inhibitors that is necessary to be used to assure a
gradual U hydrolysis in soil. This fact is very important because if the inhibitor concentration
is too high the control of ammonia volatilization may be very good but the effect of the
fertilizer on plant growth may be negative due to U accumulation in the rhizosphere.
Conversely, if the concentration of inhibitors is too low we do not get a good control of
ammonia volatilization. The case of A-soil nitrification inhibitors is quite similar to that of U-
soil hydrolysis inhibitors [49].
 The Signal Role of Nitrate in the Correction and Improvement … 79

In addition, there are potential problems derived from the eventual root uptake of the
inhibitors that can affect the metabolism of either U or A (depending on the type of inhibitor)
within the plant [50]. Taken together, these results indicate that the knowledge that we have
in order to use these types of additives, taking into account both the environmental problems
and plant growth, is very poor.
In this framework, further studies are needed in order to optimize the different techniques
that could allow us to develop N fertilizers able to assure an adequate (optimum) equilibrium
of NT, A and/or U in soil available for plants.
As we discussed above, the presence of the three N forms in soil solution, simultaneously
available for plants, has positive effects on both environmental contamination reduction
(lower NT leaching-denitrification; lower ammonia volatilization) and plant growth and
physiology improvement.
High nitrate content in conventionally produced vegetables has been observed by many
authors. This occurrence appears mainly due to fertilizers, even if environmental conditions,
such as light intensity and water availability, may affect plant nitrate and nitrite content. The
presence of these ions appears to be related to the formation of nitrosamines, characterized by
possible toxicological activity. Many studies related nitrosamine food content with cancer
risk.
We fully recognize that the content of nitrosamine present in food is higher than that
allowed by USA and EU legislation. But only few other countries have a specific legislation
for the presence of nitrosamines in foods. In the near future, health government agencies
should set the maximum permitted content of nitrosamines in foods, and provide a better
information system for consumers on how to use, transport, and store fresh food products.

ACKNOWLEDGMENT
This research was funded by CDTI, Government of Navarra and Roullier Group. Special
thanks to David Rhymes for kindly improving the English of the manuscript.

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