Annals of Applied Biology ISSN 0003-4746

RESEARCH ARTICLE

Do plants need nitrate? The mechanisms by which nitrogen

form affects plants
M. Andrews1 , J.A. Raven2,3 & P.J. Lea4
1 Faculty of Agriculture and Life Sciences, Lincoln University, Lincoln, New Zealand
2 Permanent address: Division of Plant Sciences, University of Dundee at the James Hutton Institute, Invergowrie, Dundee, UK
3 School of Plant Biology, University of Western Australia, Crawley, Australia
4 Lancaster Environment Centre, University of Lancaster, Lancaster, UK

Keywords
ABA; ammonium; ammonium toxicity;
atmospheric CO2 ; auxin; C3 plants; dry matter
partitioning; leaf expansion; low temperature
tolerance; mobilisation of seed reserves;
nitrate assimilation; nitrogen use efciency;
osmoticum; root architecture; seed dormancy.
Correspondence
M. Andrews, Faculty of Agriculture and Life
Sciences, Lincoln University, Lincoln,
New Zealand. Email:
Mitchell.Andrews@lincoln.ac.nz
Received: 2 April 2013; revised version
accepted: 24 May 2013.
Abstract
The literature on nitrogen (N) form effects on plants at different stages of their
development has been critically reviewed, assessing the possible mechanisms of
these effects. In particular, nitrate (NO3 ) was compared with the other forms
of N utilised by plants. It is concluded that the form of N available to plants
can affect their time and rate of seed germination, leaf expansion and function,
dry matter partitioning between shoot and root, and root architecture. The
magnitude of these effects is dependent on environmental factors outside the
supply of N. The mechanism of these effects is variable. Assessment of the
importance of root or shoot NO3 assimilation under different environmental
conditions is an important area for further study.

doi:10.1111/aab.12045

Introduction (Franche et al., 2009; Sprent, 2009; Andrews et al., 2011b;

Nitrogen (N) is an essential element throughout plant
growth and development as it is a constituent of DNA,
RNA, protein and hence enzymes, chlorophyll, ATP,
auxin and cytokinins (Raven et al., 2004a; Hawkesford
et al., 2012). Most plants can take up and utilise inorganic
and organic forms of N from the soil. Nitrate (NO3 ) is
the main form of N taken up and assimilated by most crop
plants in cultivated (well aerated) soils but ammonium
(NH4 + ) can be important in undisturbed/unfertilised soils
while amino acids, short peptides and even proteins
can be important in unimproved soils (Gioseffi et al.,
2012; Paungfoo-Lonhienne et al., 2012). Urea (via urine
excretion from animals or urea fertiliser) can temporarily
at least, occur in the soil at high concentrations and can
also be used by plants (Kraiser et al., 2011; Witte, 2011).
These forms of N are taken up from the soil either directly
by roots or by mycorrhizae associated with roots, and then
transferred to the plant. A small proportion of vascular
plants, in particular many legumes, all actinorhizal plants
and several C4 grasses such as sugarcane in South America
can obtain a substantial amount of their N requirements
from symbiotic microorganisms that fix atmospheric N2
Taule et al., 2012; Urquiaga et al., 2012). This ability
to access atmospheric N2 can give plants an advantage
under low soil N conditions, if other factors are favourable
for growth (Raven, 2010, 2012; Raven & Andrews, 2010).
Low soil N availability is often the major nutrient
factor limiting the growth and yield of crop plants and
the application of inorganic N fertiliser has become
an important, cost-effective strategy used to increase
crop yields in intensive agricultural systems worldwide.
However, the production and use of inorganic N fertiliser
has contributed to a range of environmental problems, in
particular, a decrease in biodiversity within and outside
the agricultural systems, emission of greenhouse gases
to the atmosphere and eutrophication of fresh waters,
estuaries, coastal waters and nutrient poor land habitats
(Andrews et al., 2011a; Butler et al., 2012). Nitrate, in
particular, is easily leached from agricultural soils and a
range of methods have been developed to reduce this
loss (Cameron et al., 2013). Generally, these methods
result in reduced levels of NO3 in soils. Also, in some
cases, for example the use of inhibitors of urease and of
nitrification, they can result in increased levels of forms
of N plants can use but which are less readily lost from

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Do plants need nitrate?
the system than NO3 (Andrews et al., 2011a; Cameron
et al., 2013).
At the plant level, N use efficiency (NUE) is dependent
on N uptake efficiency (N uptake/N available from the
soil) and N utilisation efficiency (dry matter or protein
yield/N uptake) (Andrews et al., 2004; Hirel et al., 2011;
Kant et al., 2011; Lea & Miflin, 2011; Chardon et al., 2012;
McAllister et al., 2012; Xu et al., 2012). NUE decreases with
increased N supply but it can also be dependent on the
form of N taken up and utilised (Andrews et al., 2004,
2006). Thus, to maximise NUE, a thorough understanding
of the effects of the different forms of N on crop plants
is required. This article firstly gives an overview of the
biochemistry and photon and water costs of primary N
assimilation in plants, then reviews the literature on
N form effects on plants at different stages of their
development, critically assessing the possible mechanisms
of these effects. In particular, we compare NO3 with the
other forms of N utilised by plants.
Primary N assimilation
NH4 + and NO3 assimilation
Nitrate is the most abundant source of N that is
available for plants in cultivated (well aerated) soils
with concentrations usually in the range 120 mol m3
depending on the level of N fertiliser added (Crawford
& Glass, 1998; Owen & Jones, 2001; Hawkesford et al.,
2012). Soil NH4 + concentrations are usually substantially
lower than for NO3 in cultivated soils, although they
may occur at higher concentrations than NO3 in
undisturbed/unfertilised soils (Crawford & Glass, 1998;
Owen & Jones, 2001). The available data indicate that
the mechanisms of NH4 + and NO3 uptake from the
soil by roots and their pathways of assimilation are the
same for N2 -fixing and non-fixing plants (Andrews et al.,
2004; Lea & Azevedo, 2006; Lea & Miflin, 2011). Also,
both root NH4 + and NO3 uptake are carried out by
high-affinity transport systems (HATS) and low-affinity
transport systems (LATS) (Ludewig et al., 2007; Miller
et al., 2007; Kraiser et al., 2011; Xu et al., 2012).
Evidence is strong that the integral membrane proteins
of the NH4 + transporter (AMT1) family are the major
route of high-affinity influx of NH4 + into plants (Ludewig
et al., 2007; Lanquar et al., 2009). Data for the PvAMT1;1
NH4 + transporter of common bean (Phaseolus vulgaris)
indicate that it functions as a 1:1 hydrogen ion (H+ )/NH4 +
symporter saturating at around 1 mol m3 NH4 + (Ortiz-
Ramirez et al., 2011). Earlier interpretations of plant AMT
function involved a passive NH4 + uniporter which, for a
given electrical potential across the plasmalemma, does
not allow as large an accumulation ratio (inside : outside)
as the 1H+ : 1NH4 + symporter. Structural biological
2 Ann Appl Biol (2013)
M. Andrews et al.
analysis suggests that the AMT transporters may move
ammonia (NH3 ) across the membrane, and that the
plant variants act as 1H+ : 1NH3 symporters or, at
least for PvAMT1;1, a 2H+ : 1NH3 symporter. These
stoichiometries, combined with the predominance of
NH4 + over NH3 in both the medium and cytosol,
give the overall stoichiometry for transport of NH4 + of
1H+ : 1NH4 + . The involvement of NH3 as the substrate for
transport by plant AMT transporters ensures their fidelity
and the exclusion of the potassium ion (K+ ), which
has a closely similar hydrated radius to NH4 + . Low-
affinity transport of NH4 + , which dominates at NH4 +
concentrations above 0.51 mol m3 , has been linked
to K+ transporters and non-selective cation channels
(ten Hoopen et al., 2010). NH4 + uptake is regulated
by mechanisms that act at the transcriptional and post-
transcriptional levels (Rawat et al., 1999; Yuan et al., 2007;
Lanquar et al., 2009; Rogato et al., 2010a,b).
NH4 + taken up by roots is assimilated into amino
acids via the glutamine synthetase (GS)/glutamate
synthase (GOGAT) pathway (Fig. 1; Lea & Miflin, 2011;
Hirel et al., 2011). Glutamine synthetase catalyses the
ATP-dependent conversion of glutamate and NH3 to
glutamine. Two major isoforms of GS exist; GS1 which
occurs in the cytosol of root, phloem and leaf cells
and GS2, which occurs in plastids of roots and other
non-photosynthetic tissue, and in the chloroplasts of
photosynthetic tissue. Cytosolic GSI is encoded by a
small family of 36 genes. These have been extensively
studied in a range of cereals and grasses (Swarbreck et al.,
2011), poplar (Castro-Rodrguez et al., 2011), Arabidopsis
thaliana (Arabidopsis) (Lothier et al., 2011) and legumes
(Betti et al., 2012). Initially, it was thought that there
was only one gene encoding chloroplastic GS2 in diploid
vascular plants with three in hexaploid wheat (Swarbreck
et al., 2011), however, there is now evidence of gene
duplication in Medicago truncatula (Seabra et al., 2012) and
Populus trichocarpa (Castro-Rodrguez et al., 2011).
GOGAT catalyses the nicotinamide adenine dinucleo-
tide- (NADH-) or ferredoxin- (Fd-) dependent conversion
of glutamine and 2-oxoglutarate to form two molecules
of glutamate. Both NADH- and Fd-GOGAT appear to
be solely located in plastids/chloroplasts. The NADH-
dependent GOGAT is located predominantly in non-
photosynthesising cells, where reductant is initially
supplied by the pentose phosphate pathway (Bowsher
et al., 2007). The Fd-GOGAT activity is much greater
than NADH-GOGAT in leaf chloroplasts, where light
energy is used directly for the synthesis of reduced Fd.
Compared with the information that is available for the
molecular structure of plant GS, far less is known about
GOGAT. Two genes encoding NADH-GOGAT have been
isolated from rice that are differentially expressed and
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M. Andrews et al.
Figure 1 The assimilation of nitrogen (N) in higher plants. The main enzymes involved are indicated in italics: NR = nitrate reductase; NiR = nitrite
reductase; Nase = nitrogenase; GS = glutamine synthetase; GOGAT = glutamate synthase. The ultimate source of inorganic N available to the plant is
ammonium, which is incorporated into organic molecules in the form of glutamine and glutamate through the combined action of the two enzymes
GS and GOGAT in the plastid or chloroplast. The precise route by which 2-oxoglutarate is synthesised from the tricarboxylic acid (TCA) cycle in the
mitochondria is discussed by Foyer et al. (2011).
have specific functions (Tabuchi et al., 2007; Tamura et al.,
2010), while only one has been identified in Arabidopsis
(Potel et al., 2009). Although, initially only one gene
encoding Fd-GOGAT was shown to be expressed in leaves,
there is now evidence for a second gene in Arabidopsis
and rice that is expressed at low levels throughout the
plant, in particular, in roots (Potel et al., 2009; Lu et al.,
2011).
Normally, the root is the main site of primary NH4 +
assimilation, although significant NH4 + can be detected
in the xylem sap, when plants are grown at high
NH4 + concentrations (Schjoerring et al., 2002). Also,
there is usually little accumulation of NH4 + in plant
tissues although there must be a certain steady-state
concentration of NH4 + at the site of assimilation (Raven
et al., 2008). It seems likely that GS1, and NADH-GOGAT
are involved in primary NH4 + assimilation in roots
(Andrews et al., 2004; Tabuchi et al., 2007; Masclaux-
Daubresse et al., 2010). In rice, which is normally
dependent on NH4 + when grown in paddy fields,
expression of one gene OsGS1;2 was greatly increased
in the roots by the presence of NH4 + , while OsGS1;1
was reduced. However, somewhat surprisingly, the NH4 +
content of the roots of knockout OsGS1;1 mutants was
dramatically increased, indicating that GS1;2 is unable
to substitute for GS1;1 in rice (Kusano et al., 2011).
A key role is played by chloroplastic GS2 and Fd-
GOGAT in the reassimilation of NH3 generated during
photorespiration in the leaves of C3 plants (Carvalho
et al., 2011). There is also evidence of the operation of
the photorespiratory cycle in C4 plants in the present
atmosphere, despite the accumulation of CO2 at the site
of Rubisco activity with the substantial suppression of
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Do plants need nitrate?
phosphoglycolate production. Therefore, there is still a
need for the reassimilation of NH3 generated through
the conversion of glycine to serine in the leaves of C4
plants (Lacuesta et al., 1997; Lea & Miflin, 2003). In rice,
a new form of PEP carboxylase located in the chloroplast
has been shown to play a key role in NH4 + assimilation
(Masumoto et al., 2010).
The enzyme NAD-dependent glutamate dehydroge-
nase (GDH) is also able to catalyse the incorporation of
NH3 directly into glutamate when acting in vitro. There
has been considerable debate in the scientific literature
as to whether the enzyme can operate in vivo (Dubois
et al., 2003; Labboun et al., 2009), in particular, under
stress conditions (Skopelitis et al., 2006). The properties
of mutants of Arabidopsis that lack expression of two
(Miyashita & Good, 2008), and more recently, three dif-
ferent GDH genes (Fontaine et al., 2012), now clearly
indicate that GDH operates in vivo only in the reverse
direction of glutamate metabolism, to form NH3 and
2-oxoglutarate.
Assimilation of one NH4 + molecule into plant
compounds results in the generation of 1.33 H+ which
must be excreted or neutralised to maintain cytoplasmic
pH (Raven, 1986). The bulk of H+ generated in primary
NH4 + assimilation in the root is extruded into the soil
(Raven & Smith, 1976; Andrews et al., 2004). Under
high NH4 + supply, substantial NH4 + taken up from
the soil can be transported in the xylem to the shoot
(Schjoerring et al., 2002). This is often associated with
reduced growth and, in some cases, necrotic lesions
appear in otherwise very dark green leaves. It is likely
that H+ generation by primary NH4 + assimilation in
the shoot is one of several factors that contribute to
3
Do plants need nitrate?
this damage (Britto & Kronzucker, 2002; ten Hoopen
et al., 2010; Li et al., 2012). Li et al. (2012), listed a
range of possible mechanisms to explain NH4 + toxicity;
these included disorders in pH regulation, uncoupling
of photophosphorylation, increased C consumption by
roots, futile and energetically intensive transmembrane
cycling of the NH4 + ion, and impairments in the N-
glycosylation of proteins. In particular, robust evidence
was provided that NH4 + can inhibit K+ uptake and
prevent K accumulation and redistribution (Szczerba
et al., 2008; Balkos et al., 2010; ten Hoopen et al., 2010).
However, it is unlikely that NH4 + ions will be the only
form of N that is available to plants in soils at any one
time. NO3 not only alleviates the inhibitory effect of
NH4 + , but acts synergistically to promote growth in most
but not all plant families (Pucher et al., 1947, Weissman
1964, Cox & Reisenauer, 1973; Heberer & Below, 1989;
Hecht & Mohr, 1990; Britto & Kronzucker 2002; Andrews
et al., 2009b).
Two forms of NO3 HATS have been described, a
constitutive system and an inducible system that is
stimulated by NO3 (Miller et al., 2007; Kraiser et al., 2011;
Xu et al., 2012). These systems are the main route of NO3
uptake into the plant at low soil NO3 concentrations
(<0.51 mol m3 ). The NO3 LATS becomes significant
at external NO3 concentrations above 1 mol m3 (Miller
et al., 2007; Glass, 2009). For Arabidopsis, root NO3
uptake is primarily carried out by the NRT1 and NRT2
families of NO3 transporters (Dechorgnat et al., 2011)
via a 2H+ /1NO3 symport driven by a transmembrane
pH and electrical potential gradients (Miller et al., 2007;
Kraiser et al., 2011).
There are 53 members of the NRT1 gene family in
Arabidopsis of which NRT1-1 has been the most extensively
characterised (Tsay et al., 2007). The NRT1.1 (CHL1)
transporter in Arabidopsis can function in the HATS when
phosphorylated and LATS when dephosphorylated (Liu
& Tsay, 2003). In contrast to NRT1, there are only seven
genes in the NRT2 family in Arabidopsis. The NO3
inducible HATS system is carried out predominantly
by NRT2.1 in collaboration with NAR2.1 in Arabidopsis
(Kotur et al., 2012), while NRT2.4 is active even following
N starvation (Kiba et al., 2012). In rice, four NRT2 and
two NAR2 genes encode HATS components (Feng et al.,
2011). The spatial variability of NO3 uptake along the
maize primary root axis has been investigated at the
physiological and molecular level with respect to NRT2.1
(Sorgona et al., 2011).
Nitrate taken up by the roots can be stored or
assimilated in the roots or transported in the xylem to
the shoot, where again it can be stored or assimilated.
Normally, there is little transport of NO3 in the phloem
but it does occur and in Arabidopsis is mediated by the
4 Ann Appl Biol (2013)
M. Andrews et al.
NRT1.7 and NRT1.9 NO3 transporters (Wang et al.,
2012b). Vacuoles are the main site of NO3 storage
in roots and shoots (Hawkesford et al., 2012; White,
2012). Storage of NO3 is dependent on plant genotype,
plant tissue and environmental conditions. Some leaf
crops such as lettuce and spinach typically accumulate
substantial NO3 (Sindelar & Milkowski, 2012). Due
to fears that green leaf consumption could result in
excessive NO3 intake, the EU has imposed legally
binding maximum limits on the NO3 concentrations
in marketed products (Burns et al., 2012).
Nitrate assimilation in plants, initially involves reduc-
tion to NH4 + which is followed by assimilation via the
GS/GOGAT pathway in the same way as NH4 + taken
up from the soil (Fig. 1). The first step of NO3 reduc-
tion requires the enzyme nitrate reductase (NR), which
converts NO3 to NO2 , which is followed by the reduc-
tion of NO2 to NH4 + by nitrite reductase (NiR) (Lillo,
2008; Heidari et al., 2011). Nitrate reductase is located
in the cytosol of root and shoot cells and for most plant
species the major NR isoform involved in NO3 assimi-
lation uses NADH as the reductant. The activity of the
enzyme is subject to regulation at the level of gene
expression and by reversible phosphorylation and the
interaction with 14-3-3 proteins (Campbell, 2002; Lillo,
2008; Heidari et al., 2011; Lambeck et al., 2012). The
NiR enzyme is located in the plastids of roots and other
non-photosynthetic tissue and the chloroplasts of photo-
synthetic tissue and uses Fd as a reductant (Sakakibara
et al., 2012). Synthesis of NiR is regulated by a NO3
responsive cis-element (Konishi & Yanagisawa, 2011) but
there is no evidence of post-translational modification
(Campbell, 2002). Generally, NR and NiR are substrate
and light induced enzymes (Lillo, 2008), although there
are high levels of constitutive NR in the leaves of some
members of the Phaseoleae (Andrews et al., 1990) and
on rare occasions, NR is induced by NH4 + or urea
(Bungard et al., 1999; Matraszek, 2008; Garnica et al.,
2010).
The root or shoot can be the main site of NO3
assimilation depending on genotype and environmental
conditions, especially soil NO3 concentrations over
the range found in agricultural soils. For example,
for temperate cereals and grain legumes, the root is
the main site of NO3 assimilation at external NO3
concentrations around 1 mol m3 or less, but shoot NO3
assimilation increases in importance as the external
NO3 concentration increases in the range 120 mol m3
(Andrews et al., 1992, 2004). In contrast, some tropical
and sub-tropical cereals and grain legumes carry out
the major proportion of their NO3 assimilation in the
shoot, whether growing in low or high external NO3
concentrations (Andrews et al., 2004).
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M. Andrews et al.
The assimilation of one molecule of NO3 into plant
organic compounds results in the generation of 0.67
molecules of OH (Raven, 1985). Much of the OH
generated in root NO3 assimilation is extruded into
the soil, while the major proportion of OH generated
in shoot assimilation is neutralised by the synthesis of
organic acids, in particular, malate. Often, this malate
is retained in the shoots although some species have the
capacity to co-transport malate and K+ from shoot to root,
which is linked to OH excretion from the root (Andrews
et al., 2004; Peuke, 2010). High NO3 supply, as for NH4 + ,
can inhibit growth but with NO3 the visual symptoms
are generally inter-veinal chlorosis caused by iron (Fe)
deficiency. Iron has extremely low solubility at neutral or
basic pH and there is evidence that the increased pH at
the root surface with NO3 nutrition inhibits Fe uptake
(Nikolic & Romheld, 2003; Zhao & Ling, 2007). There
is also evidence that increased pH in the apoplast due to
NO3 assimilation inactivates Fe in leaf apoplasts (Mengel
& Geurtzen, 1988; Kosegarten et al., 1999; Zhao & Ling,
2007).
Urea assimilation
Urea-N inputs into agricultural systems are substantial.
Urea is the main form of fertiliser N applied globally, and
its concentration can be as high as 700 mol m3 in animal
urine (Witte, 2011; Cameron et al., 2013). Nevertheless,
little work has been carried out to assess the importance
of direct uptake and assimilation of urea by plants in
agricultural systems (Witte, 2011). For Arabidopsis and
rice, respectively, the AtDUR3 and OsDUR3 high-affinity
urea transporters have been characterised (Kojima et al.,
2007; Wang et al., 2012a). The mechanism of urea uptake
by the AtDUR3 transporter for Arabidopsis is H+ /urea co-
transport (Kojima et al., 2006, 2007). By analogy with
the Na+ /urea transport in the charophycean freshwater
green alga Chara corallina (Walker et al., 1993), it is likely
that the H+ /urea ratio in plant roots is 1:1. Expression
of the AtDUR3 gene in N deficient Arabidopsis roots was
induced after the supply of urea but repressed by NH4 +
and NO3 (Kojima et al., 2006, 2007; Merigout et al.,
2008). There is evidence for Arabidopsis and maize that
low-affinity uptake of urea is mediated by aquaporins
(Kojima et al., 2006, 2007; Gu et al., 2012). However,
kinetic evidence from C. corallina is consistent with low-
affinity uptake of urea occurring by a slip mechanism
which allows the high-affinity Na+ /urea symporter to
function as a low-affinity urea uniporter (Walker et al.,
1993).
Within the plant, urea undergoes hydrolysis via the
enzyme urease with NH3 and CO2 being produced (Witte,
2011). The NH3 is assimilated into amino acids via
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Do plants need nitrate?
the GS/GOGAT pathway as described above. There is
evidence for common bean that the root is the main site
of urea assimilation at 12 mol m3 applied urea-N but
shoot assimilation increases in importance as the applied
urea-N concentration increases from 2 to 10 mol m3
(Hine & Sprent, 1988). Data for partitioning of urea
assimilation between root and shoot of other species
are not available. The assimilation of urea results in the
production of 0.33H+ per N assimilated (Raven, 1986).
Some plants reliant on urea as the sole N source exhibit
limited growth (Andrews et al., 2006; Merigout et al.,
2008; Witte, 2011). As for NH4 + , NO3 can alleviate
the inhibiting effect of urea, and in some cases act
synergistically to promote growth (Witte, 2011).
Nitrogen fixation
Root nodules are the primary site of N2 fixation
for legume and actinorhizal crops. Atmospheric N2 is
reduced to NH3 via the bacterial enzyme nitrogenase in
rhizobial bacteroids in legumes and Frankia endophytes
in actinorhizal plants (Franche et al., 2009; Sprent, 2009).
The overall reaction is:
N2 + 8H+ + 8e + 16ATP 2NH3 + H2 + 16ADP + 16Pi
For some legume species, the H2 is taken up by an uptake
hydrogenase and used in oxidative phosphorylation (Den
Herder & Parniske, 2009; Golding & Dong, 2010). There
is a net production of 0.33H+ per unit N fixed into plant
compounds via N2 fixation. The evidence is consistent for
legumes that the bulk of the H+ generated in N2 fixation
is extruded into the soil (Raven, 1985; Andrews et al.,
2009a).
Generally, NH4 + produced via N2 fixation is trans-
ported from the rhizobial bacteroids and Frankia endo-
phytes to the plant root cells where it is assimilated into
amino acids via the GS/GOGAT pathway (Betti et al.,
2012; Seabra et al., 2012). However, evidence is strong
that Frankia carries out both N2 fixation and the biosyn-
thesis of arginine within the nodules of Datisca glomerata
(Berry et al., 2011). It has also been suggested that ala-
nine is a major transport compound from the bacteroids
in soybean, possibly through the action of alanine dehy-
drogenase (Waters et al., 1998), but this has not been
confirmed (White et al., 2007; Mulley et al., 2011). For
most N2 fixing crops, the amino acid amides glutamine
and asparagine are the main N containing compounds
derived from N2 fixation, which are transported from
root to shoot (Lea et al., 2007), but there are exceptions.
In particular, for legume species in the tribes Desmodiae,
Psoraleae and the Phaseoleae, which includes important
crop species such as soybean, common bean and cowpea,
the ureides allantoin and allantoic acid are the main N
5
Do plants need nitrate?
containing compounds transported from root to shoot in
N2 fixing plants (do Amarante et al., 2006; Todd et al.,
2006; Sprent, 2009). However, when these species are
grown on NO3 without nodules, N is primarily trans-
ported from root to shoot as NO3 and amino acids (do
Amarante et al., 2006). Nevertheless, ureides may also be
used for N transport in NO3 fertilised plants, especially
at pod filling (Thomas et al., 1980; Daz-Leal et al., 2012).
Data suggest that remobilised N from lower leaves is the
source of these ureides (Daz-Leal et al., 2012). In the root
nodules of the FrankiaAlnus incana symbiosis, citrulline
plays a major role in N assimilation (Schubert, 1986;
Lundquist et al., 2003; Lundberg & Lundquist, 2004).
In the shoot, the ureides and citrulline are metabolised
and NH3 is released and subsequently reassimilated via
GS/GOGAT in secondary assimilation (Fig. 1; Werner &
Witte, 2011; Duran & Todd, 2012).
Photon and water costs
Andrews et al. (2009a) updating the work of Raven
(1985), carried out a cost benefit analysis based on
biochemical principles of the photon and water costs of
the various biochemical and transport processes involved
in plant growth, N assimilation, pH regulation and
osmolarity generation, per unit N assimilated with NH4 + ,
NO3 or N2 as N source. These data are shown in Table
1 along with additional data for 1H+ : 1NH4 + symport
and urea entry by passive urea uniport and 1H+ : 1 urea
symport, estimated as in Andrews et al. (2009a). For the
newly calculated data, NH4 + assimilation after entry by
1H+ : 1NH4 + symport has energy and water costs only
1% more than the corresponding values for NH4 + entry
by passive uniport. For urea uptake and assimilation,
the energy and water costs for passive urea uniport are
less than 1% lower than the value for NH4 + entry by
passive uniport. The same applies to the comparison of
urea uptake by 1H+ : 1 urea symport and 1H+ : 1NH4 +
symport. These differences between urea and NH4 + are
the result of urea entry and assimilation being, in charge
and H+ production terms, equivalent to the entry of NH3 ,
so that in each case there is an energy and water saving
corresponding to the active efflux of 1H+ when compared
with the corresponding uptake of NH4 + .
Across N forms, the predicted values for photon and
water costs were lower for NH4 + and urea assimilation,
than for NO3 assimilation and N2 fixation (Table 1).
Values for photon and water costs were respectively
5.311.7% and 2.411.1% greater for NO3 assimilation
than NH4 + or urea assimilation depending on the site of
NO3 assimilation (root or shoot) and the mechanism of
associated pH regulation. The photon and water costs
were respectively 7.810.6% and 8.310.7% greater
6 Ann Appl Biol (2013)
M. Andrews et al.
for N2 fixation than for NH4 + or urea assimilation.
Thus photon and water costs can be greater or less for
NO3 assimilation than for N2 fixation depending on
the site of assimilation and mechanism of pH regulation.
Overall, photon and water costs per unit N assimilated
are respectively likely to be around 5 and 7% greater
for N2 fixing crops than for non-N2 -fixing crops relying
on a combination of NH4 + and root and shoot NO3
assimilation (Andrews et al., 2009a). It is emphasised that
the predictions of photon and water use as a function
of N source are for optimally allocating plants, those
which use their resources in a way which maximises
the acquisition and assimilation of other resources under
the prevailing conditions (Rosen, 1967), such that they
minimise their energy and water use. In some cases,
these predictions do not agree with experimental findings
(Raven et al., 2004a,b). In particular, for a number of
dicotyledonous crops across different studies, the water
lost per unit dry matter gain was greater for plants
grown in NH4 + than on NO3 . At least part of this
effect could have been due to slower growth with NH4 +
as a N source (Raven et al., 2004a,b). Also, the water
and energy cost analysis performed deals only with
the running costs of N acquisition and assimilation.
There are also construction and maintenance costs which
differ among N forms and assimilation processes. The
most obvious of these are the root nodules which
only occur and function in symbiotically diazotrophic
plants. However, any additional costs associated with the
construction and maintenance of root nodules must be
balanced against a decreased requirement for roots as
scavengers of combined N in symbiotic diazotrophs, with
the complication that nodule-free root systems, unlike
nodules, function in the uptake of other nutrient elements
and water, and are required for anchorage.
For some species grown on high NO3 supply or under
conditions in which photosynthesis is reduced, such as
low irradiance, a substantial proportion of the NO3
taken up is not assimilated. Under these conditions,
NO3 can be an important osmoticum in tissues, taking
the place of organic molecules, in particular, hexose
sugars (mainly glucose) and organic acids (mainly malate)
(Raven et al., 1980; Andrews et al., 2005; Hummel et al.,
2010). Andrews et al. (2005) calculated the energy cost
of osmoticum as KNO3 as 11 mol photons osmol1 which
was less than half that for K2 -malate (24 mol photons
osmol1 ), and less than one-seventh that for hexose
(80 mol photons osmol1 ). Thus, the use of KNO3 as an
osmoticum can be a significant energetic advantage.
Recent work has shown that under anoxic conditions,
mitochondria isolated from barley and rice roots retain
a limited capacity to oxidise external NADH/NADPH
and synthesise ATP linked to the reduction of NO2
2013 Association of Applied Biologists
M. Andrews et al. Do plants need nitrate?

Table 1 Nitrogen form effects on the photon and water costs for overall plant growth expressed on a plant organic N basis

Photon Cost (mol H2 O Cost

absorbed photons (mol H2 O
Site/Mechanism of Assimilation mol1 plant transpired mol1
and Associated pH Regulation organic N) plant organic N)

NH4 + assimilation in root, with NH4 + entry by electrically driven uniport, 371 16 000
H+ excreted from root
NH4 + assimilation in root with NH4 + entry by 1H+ : 1NH4 + symport, H+ 374 16 118
excreted from root
Urea assimilation following urea entry by passive uniport 368 15 882
Urea assimilation following urea entry by 1H+ : 1 urea symport 371 16 000
NO3 reduction/assimilation in root with
(a) OH- excreted from roots 397 16 658
(b) OH- disposal by organic acid synthesis and accumulation of organic 400411 17 45017 640
anions in the root cells or excretion of organic anions to the medium
NO3 photoreduction/assimilation in shoot with
(a) OH- disposal by oxalic acid synthesis and oxalate2 accumulation in 401 16 540
shoot cell vacuoles
(b) OH disposal by malic acid synthesis and malate2 accumulation in 407 16 550
shoot cell vacuoles
(c) OH disposal by malic acid synthesis, malate transfer to the root with 394 16 510
malate catabolism and OH excretion to the root medium
N2 xation in root nodules, H+ excreted from root; no H2 consumed by 407 17 585
uptake hydrogenase
N2 xation in root nodules, H+ excreted from root; all H2 consumed by 403 17 450
uptake hydrogenase

to NO in the mitochondrial electron transport chain
(Stoimenova et al., 2007; Igamberdiev & Hill, 2009; Gupta
& Igamberdiev, 2011). The importance of these reactions
in relation to vascular plant tolerance of anoxic or hypoxic
conditions has yet to be assessed.
Seed germination
Dormancy
Seed dormancy is a mechanism utilised by plants to
maximise the chances that seed germination occurs under
conditions that are suitable for continued plant survival
and growth (Finkelstein et al., 2008). There is considerable
evidence that the levels of the plant hormone abscisic acid
(ABA) in the seed, play an important role in the regulation
of seed dormancy and germination (Finkelstein et al.,
2008; Nambara et al., 2010; Footitt et al., 2011). Normally,
release from dormancy is associated with a decrease in
ABA and an increase in gibberellic acid (GA), a hormone
that promotes seed germination (Rajjou et al., 2012).
Environmental factors, in particular, low temperature,
light and NO3 , can individually and interactively reduce
seed dormancy of many species (Gallagher & Fuerst, 2006;
Hilhorst et al., 2006; Andrews et al., 2009b; Finch-Savage
& Footitt, 2012). Ammonium, urea and amino acids,
can reduce seed dormancy of some species (Hendricks
& Taylorson, 1974; Goudey et al., 1987; Bungard et al.,
1997; Alboresi et al., 2005; Atia et al., 2009) but this effect
is much less common than with NO3 . The mechanism
of the NO3 specific effect on seed dormancy is not fully
understood for any one species. Most recent detailed
work in this area has been carried out on Arabidopsis
and this is focused on here but it is acknowledged that
NO3 could act on the seed dormancy of other species
in different ways (Finkelstein et al., 2008). Studies of
whole and isolated parts of Arabidopsis seeds indicated
that the aleurone layer was the primary determinant of
seed dormancy but embryos from dormant seeds had a
lower growth potential than those of non-dormant seeds
(Bethke et al., 2007). Also, several studies have shown
that the seed dormancy of Arabidopsis is lost during dry
storage (Matakiadis et al., 2009).
Application of 10 mol m3 N as NO3 but not as NH4 +
or glutamine to a germination medium, reduced the
seed dormancy of Arabidopsis (Alboresi et al., 2005).
In the same study, the germination of mutants with
low levels of the NO3 transporter NRT1.1, was not
stimulated by 1 mol m3 NO3 , while the dormancy of
the wild type was reduced. Increased NO3 application
from 3 to 10 mol m3 or 10 to 50 mol m3 to the
mother plant during seed development also reduced the
dormancy of the seed. In addition, seeds of mutants with
greatly reduced NR activity showed reduced dormancy
in comparison with the wild type. Overall, there was

Ann Appl Biol (2013) 7

2013 Association of Applied Biologists
Do plants need nitrate?
a strong negative correlation between NO3 levels in
seeds and dormancy and it was concluded that NO3 is a
signal relieving seed dormancy in Arabidopsis. Matakiadis
et al. (2009) extended this study and showed that the
decreased dormancy associated with increased NO3
supply in the germination medium or to the mother
plant during seed development, was associated with
lower concentrations of ABA in the imbibed seeds. For
the wild type plants, lower concentrations of ABA were
correlated with increased expression of the ABA catabolic
gene CYP707A2, which encodes ABA 8 -hydroxylase in
seeds. Expression of CYP707A2 was also induced in the
vegetative tissue of NH4 + fed plants when supplied NO3 ,
indicating that it is a NO3 responsive gene. These results
indicated that NO3 induction of CYP707A2 expression
and hence metabolism of ABA could be an important
mechanism of NO3 diminution of seed dormancy of
Arabidopsis.
Bethke et al. (2006a,b) linked the NO3 effect on
seed dormancy of Arabidopsis to the production of nitric
oxide (NO) as the NO scavenger 2-(4-carboxyphenyl)-
4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO)
maintained seed dormancy in NO3 treated plants. Liu
et al. (2009) reported that a rapid accumulation of NO in
Arabidopsis seed over the 48 hours after treatment with
the NO donors sodium nitroprusside and S-nitroso-N-
acetyl-DL-penicillamine (SNAP) induced an equally rapid
decrease of ABA which was correlated with increased
CYP707A2 transcription, synthesis of ABA 8 -hydroxylase
and reduced dormancy. In contrast, in the cyp707a2
mutant with decreased ABA catabolic activity, ABA levels
and dormancy changed little in response to the NO
donors. These results indicate that the CYP707A2 protein
played a crucial role in the rapid decrease of ABA and
dormancy after 48 hours imbibition of the NO treated
wild type seed. Thus, linking the studies of Matakiadis
et al. (2009), Bethke et al. (2006a,b) and Liu et al. (2009),
NO3 induced CYP707A2 expression in Arabidopsis may be
related to the generation of NO. Nitric oxide reduces seed
dormancy in many species that respond to NO3 (Bethke
et al., 2006a). Thus NO3 may act on seed dormancy
of Arabidopsis and other species, at least in part, via the
production of NO and its subsequent effects on levels
of ABA in seeds but this needs further work including
determination of how NO is produced from NO3 in
seeds. Nitric oxide can be produced in plants in several
ways including the NAD(P)H-dependent reduction of
nitrite (NO2 ) to NO by NR (Gupta et al., 2011). However,
the finding that NO3 accumulation reduces dormancy
in the seeds of Arabidopsis NR mutants indicates that
for this species, levels of NR over a wide range are not
a factor in the NO3 effect on dormancy (Finkelstein
et al., 2008). With regard to agricultural systems, seed
8 Ann Appl Biol (2013)
M. Andrews et al.
dormancy has been selected against for many crop species
with the objective of increasing the rate and uniformity
of germination (Finkelstein et al., 2008). As a result, seed
dormancy and its reduction by NO3 are of much greater
importance to wild plants than crop species.
Mobilisation of seed reserves
At concentrations of 520 mol m3 N, NO3 but not
NH4 + or urea increased the rate of mobilisation of seed
reserves of non-dormant seeds of wheat, barley, oat,
rye and triticale prior to emergence from the substrate
(Lieffering et al., 1996; McIntyre, 2001). Application of
5 mol m3 chloride (Cl ) had a similar effect to NO3 on
mobilisation of the seed reserves of barley (Lieffering
et al., 1996). Increased rates of mobilisation of seed
reserves with additional NO3 or Cl were associated
with increases in shoot, root and residual seed anion
content, total seedling water and residual seed water
content, 21 days after sowing. Addition of NH4 + did not
affect total seedling water or residual seed water content.
Nitrate and Cl comprised around 70% of the total anions
measured in seedlings supplied 5 mol m3 NO3 and
Cl , respectively. For seedlings supplied with NO3 , NR
activity was low in the roots and shoots, which is likely
to have been a factor causing NO3 accumulation in the
seedlings. Total osmoticum was around 30% greater with
the addition of 5 mol m3 NO3 and total anion content
with counter ions constituted around 20% of the total
solute determined for barley roots and shoots. For barley
seeds supplied with different concentrations of NO3 or
mannitol, the rate of mobilisation of seed reserves was
positively correlated (r > 0.95) with total seedling water
and residual seed water content. These findings provide
evidence that the increased rate of mobilisation of seed
reserves of temperate cereals induced by additional NO3
was due to increased water uptake as a result of increased
NO3 uptake and accumulation.
Lieffering et al. (1996) argued that the seedling root,
not the seed, was the primary site of NO3 and increased
water uptake. However, the possibility that substantial
NO3 and its counterions (mainly K+ ) can be taken up
directly by the seed is worth testing as Zhang et al. (2010)
have shown that barley seeds can take up Na+ (and
most likely Cl ) directly, to osmotically important levels.
Evidence was presented that under saline conditions,
this Na+ (Cl ) allowed the plant to generate additional
osmotic potential and hence absorb more H2 O and
germinate more rapidly in environments of low water
potential.
The rate of mobilisation of seed reserves of temperate
cereals is dependent on NO3 concentration over the
range likely to occur in agricultural soils (Lieffering et al.,
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M. Andrews et al.
1996). Thus reserve mobilisation could be affected by
different management practices that influence soil NO3
levels such as no tillage, cultivation of soil and use of N
fertiliser (Cameron et al., 2013). These possibilities need
to be tested. It is also not known if NO3 increases the
rate of mobilisation of seed reserves of other crop species.
Leaf expansion and function
Leaf expansion
Often, at the crop and plant level, leaf area, radiation
interception, photosynthesis and dry matter production
are all positively correlated (Monteith, 1977; Adam et al.,
2011; Duursma et al., 2011; Massignam et al., 2011). Thus,
the extent of leaf expansion is a major factor in crop/plant
performance. Pantin et al. (2011, 2012) argued that water
and C are the major factors limiting leaf expansion. Water
acts as a biophysical control mainly linked to water fluxes
to growing cells and C is a metabolic control linked
to the supply of carbohydrates for energy and structural
components. Carbon is also required for the production of
organic molecules used to maintain the osmotic potential
of the cell (Raven et al., 1980; Andrews et al., 2005;
Hummel et al., 2010). There is considerable evidence that
cell osmotic potential plays a central role in cell expansion
by generating turgor pressure, which ultimately stretches
cell walls (Lockhart, 1965; Pantin et al., 2011, 2012).
The growth of many plant species is greater with NO3
than with NH4 + as a N source, as high concentrations
of NH4 + can cause NH4 + toxicity. However, Walch-Liu
et al. (2000) reported that for two cultivars of tobacco,
shoot and root growth were substantially greater with
NO3 than NH4 + supplied over periods of 1014 days,
even at low (1.52.5 mol m3 ) N application rates. Leaf
number was similar for plants grown on the two N forms
but leaf expansion was greatly reduced in plants grown
on NH4 + due to a reduction in cell number and size.
The concentrations of N in shoot tissues were similar
with NO3 and NH4 + as individual N sources, while the
concentrations of sugars and starch were greater with
NH4 + . Concentrations of the macronutrient cations K+ ,
magnesium (Mg2+ ) and calcium (Ca2+ ) in the press sap
were similar for the young leaves of plants grown on
either NH4 + or NO3 , but for mature leaves were 3050%
lower with NH4 + . Nitrate occurred at concentrations of
2338 mol m3 in the press sap of leaves of NO3 fed
plants but was undetectable in the leaves grown on
NH4 + . A deficiency of NO3 as an osmoticum in NH4 +
fed plants was compensated for by an increase in Cl
concentration. Similarly, the osmotic potential of press
sap of young leaves was not affected by N form but
was less negative in the older leaves of plants grown on
NH4 + .
Ann Appl Biol (2013)
2013 Association of Applied Biologists
Do plants need nitrate?
A switch from NO3 to NH4 + as N source caused a
reduction in the rate of leaf expansion of tobacco within
24 h, while re-application of NO3 caused an equally
rapid increase in leaf expansion (Walch-Liu et al., 2000).
For plants supplied with NH4 NO3 as a N source, transfer
to NO3 or NH4 + did not affect the apparent hydraulic
conductivity of the roots 1 or 5 days after transfer,
or water uptake over 2 days. However, within 24 h of
transfer to NH4 + , the xylem sap NO3 content decreased
dramatically, by 40-fold, while the zeatin and zeatin
riboside fraction of cytokinins in the xylem sap decreased
by 70%. It was concluded that the rapid inhibition of leaf
growth by NH4 + was not due to limited availability of N,
carbohydrate, mineral nutrients, osmoticum or water but
to a lack of NO3 supply. It was also proposed that the
presence of NO3 is required to maintain the biosynthesis
of cytokinins and/or their root to shoot transfer at a level
that is sufficient to mediate normal leaf morphogenesis.
However, the finding of considerably decreased levels of
macronutrient cations (K+ , Mg2+ , Ca2+ ) in the mature
leaves of NH4 + fed plants indicates that uptake of these
ions was reduced with NH4 + and that their levels were
being maintained in young leaves by re-translocation
from older leaves. A lower rate of uptake and greater re-
translocation of K+ from mature to expanding leaves of
plants grown in low concentrations of NH4 + as compared
to NO3 , was confirmed in tobacco (Lu et al., 2005). Thus
a reduced uptake of macronutrient cations could play a
major role in reduced leaf expansion with NH4 + .
Common bean is similar to tobacco in that it transports
substantial NO3 to the shoot when grown on a high
NO3 supply, and the shoot is then the main site of
NO3 assimilation. The expansion rate of common bean
leaves is very responsive to NO3 supply and growth
is considerably greater with NO3 than with NH4 + as a
N source (Sprent & Thomas, 1984; Zerihun et al., 1998;
Andrews et al., 2001, 2004). However, the total plant
leaf area and dry matter production achieved by common
bean can be as great when grown on forms of N other
than NO3 (Zerihun et al., 1998; Andrews et al., 2001,
2004). Focusing on leaf expansion, total plant leaf area
achieved was twice as great with NO3 than NH4 + but
similar with NO3 , urea, glutamine and allantoic acid
(Fig. 2a). These results emphasise that maximum leaf
area can be achieved with N nutrition other than NO3 .
Maximum leaf area was achieved at 6 mol m3 N as NO3
but at 10 mol m3 N as urea, allantoic acid or glutamine.
Also, the maximum area was achieved with less leaf N
with NO3 , in comparison to the other N forms (Fig. 2
b). Maximum dry matter production was also achieved
with less total plant N with NO3 , in comparison with the
other N forms (Zerihun et al., 1998; Andrews et al., 2001,
2004). This finding does not match theoretical predictions
9
Do plants need nitrate?
in relation to NO3 versus urea as a N source (Table 1).
The greater leaf area per unit N with NO3 could be
related to greater NO3 transport to, and assimilation
in the shoot leading to greater osmoticum supply, and
hence greater leaf expansion (Sprent & Thomas, 1984).
The finding of greater K+ and ash alkalinity per unit
area with NO3 , rather than with urea, glutamine and
allantoic acid (Fig. 2c and Fig. 2d) is consistent with this
proposal and this warrants further testing. It is likely that
this effect could be mimicked in crop plants which reduce
most of their NO3 in the root, by selection of plants
which express NR and NiR primarily in the shoot and this
could increase plant NUE (Andrews et al., 2004).
The NH4 + toxicity problem is now relatively rare in
agricultural systems. However, N inputs are required
for most crops to maintain the leaf expansion rate and
achieve maximum leaf area due to the removal of N in
harvested plant products and hence depletion of soil N.
Greater reliance on forms of N other than NO3 - could
reduce plant NUE.
Stomatal function
Stomata play a major role in leaf function by controlling
CO2 uptake and H2 O loss via changes in stomatal aperture
(Pantin et al., 2012). Stomatal aperture is regulated
by turgor driven expansion and contraction of guard
cells. The NO3 transporter gene AtNRT1.1 is expressed
and functions in Arabidopsis guard cells (Guo et al.,
2003). On a high NO3 /low Cl medium, the Atnrt1.1
mutant accumulated less NO3 in the guard cells during
stomatal opening and failed to show NO3 induced
depolarisation of guard cells. These differences in the
mutant in comparison with the WT were reflected in
reduced stomatal aperture and reduced transpiration
rates in the light or when deprived of CO2 in the
dark. It was suggested that NO3 transport mediated
by AtNRT1.1 functions to regulate stomatal aperture.
Kanno et al. (2012) demonstrated that the previously
identified NO3 transporter NRT1.2 is able to act as an
ABA transporter mediating cellular uptake. Therefore, the
precise interaction between NO3 and ABA in stomatal
function is still not clear. Currently, there is no evidence
that NO3 is a pre-requisite for stomatal function (Guo
et al., 2003; Lamattina & Garca-Mata, 2003; Raven, 2003;
Kanno et al., 2012).
Dry matter partitioning between shoot and root
Nitrogen supply, regardless of form, has a strong and
consistent effect on dry matter partitioning between the
shoot and root of vascular plants from the seedling
stage to maturity. Specifically, the shoot to root dry
10
M. Andrews et al.
weight ratio (S : R) increases with increased N supply
over the range likely to occur in natural and agricultural
soils (Andrews et al., 1999, 2001; Poorter et al., 2012).
However, there are many reports that at similar total
plant dry weights, the S : R is dependent on the form of
N nutrition with comparisons involving two or more of
NO3 , NH4 + , urea, N2 and amino acids (Andrews et al.,
1999, 2001, 2006; Cambui et al., 2011). Work on mutants
and transgenic lines of tobacco with decreased expression
of NR demonstrated a highly significant, linear positive
correlation between S : R and leaf NO3 content for eight
different genotypes growing on a wide range of NO3
concentrations. It was proposed that NO3 acts as a signal
to regulate dry matter partitioning between shoot and
root of vascular plants (Scheible et al., 1997).
Andrews et al. (2006) challenged the hypothesis
that NO3 acts as a signal to regulate S : R. It was
highlighted that in the study of Scheible et al. (1997),
most of the change in S : R of tobacco was associated
with exceptionally high leaf NO3 concentrations,
which would rarely occur under natural or agricultural
conditions. Values for S : R ranged from around 210,
with those above 3.5 associated with leaf NO3
concentrations of 5003000 mol g1 dry weight. Also,
there were deviations from the strong relationship
between S : R and leaf NO3 at low leaf NO3
concentrations. Plants that were grown on low NO3
had S : R values that lay below the regression line, while
plants grown on NH4 + alone or NH4 NO3 had S : R values
above the regression line. In addition, the relationship
between S : R and leaf NO3 content did not hold under
phosphorus (P) deficiency, or at double the ambient CO2
concentration (Scheible et al., 1997; Kruse et al., 2002).
Andrews et al. (2006) argued on the basis of results
from the literature and experimental data for tobacco
that N (concentration and form) and other environmental
influences on S : R are often primarily mediated through
effects on leaf protein concentration and hence shoot and
then plant growth. For tobacco grown under a range
of N forms, macronutrient deficiencies and irradiance
levels, S : R across all treatments was not significantly
correlated with leaf NO3 concentration, but there was a
strong positive relationship between S : R and leaf soluble
protein concentration. It was proposed that normally,
shoot growth is co-limited by the availability of C
and N substrates and that the shoot soluble protein
concentration is of particular importance as this reflects
the availability of the N substrate for shoot growth.
Thus, the increase in S : R observed with increased leaf
soluble protein concentration across a wide range of
environmental conditions, may be due to an increase
in N relative to the C substrate for shoot growth. The
proximity of the shoot to the C and energy sources
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2013 Association of Applied Biologists
M. Andrews et al. Do plants need nitrate?

(a) (b)
400 400

Whole plant leaf area (cm 2 )

Whole plant leaf area (cm 2 )

300 300

200 200

Aln
100 Gln 100
NH4+
NO3-
Urea
0 0
0 2 4 6 8 10 0 2 4 6 8 10 12 14
-2
Applied N (mol m-3 ) Leaf N concentration (mol cm )

(c) 5 (d) 6
Leaf K concentration (mol cm-2 )

Leaf ash alkalinity (eq cm )

-2
4 5

3 4

2 3

1 2
0 0
0 2 4 6 8 10 0 2 4 6 8 10
Applied N (mol m-3 ) Applied N (mol m-3 )

Figure 2 Leaf area (a), leaf area per unit nitrogen (b), leaf potassium concentration (c) and leaf ash alkalinity concentration (d) of Phaseolus vulgaris
supplied different concentrations of allantoin (Aln, ), glutamine (Gln, ), ammonium (NH4 + , ), nitrate (NO3 , ) and urea ( ). Modied from Zerihun
(1996).

results in the shoots acquiring an increased proportion Root architecture

of photosynthate for growth, if the supply of N substrate
The architecture of plant roots can be changed by
increases relative to the C substrate for growth. The
the availability of nutrients (Robinson, 1994; Forde &
greater the proportion of photosynthate utilised in shoot
Lorenzo, 2001; Hodge et al. 2009; Smith & de Smet,
growth, the smaller the proportion available for transport
2012) and there is evidence that plants can forage in
to the root, and, as a result, the S : R increases.
nutrient rich patches (Crick & Grime, 1987; de Kroon
Two studies since Andrews et al. (2006) have argued
et al., 2009). Early experiments indicated that locally
that S : R is dependent on N form (Markham & Zekveld, applied NO3 is able to stimulate lateral root growth
2007; Cambui et al., 2011) but neither tested the in pea (Wiersum, 1958), wheat (Hackett, 1972), barley
relationship between S : R and leaf soluble protein (Drew et al., 1973) and maize (Granato & Raper, 1989).
concentration. It is emphasised that in other studies, The classical experiments of Drew and colleagues with
S : R was strongly correlated with leaf soluble protein barley, which are reviewed in Drew (1975), demonstrated
concentration across N forms (Andrews et al., 2006). an increased number and length of lateral roots induced
Overall, it is our view that considerable data in the by the localized application of NO3 , NH4 + and P, but
literature indicate that the effects of different N forms not K.
on S : R for a given total plant dry weight, are mediated Using Arabidopsis grown on sterile segmented agar
through leaf soluble protein concentration. Greater plates, Zhang & Forde (1998) and Zhang et al. (1999)
allocation of resources to roots under N limitation is showed that locally applied NO3 patches increased the
likely to be an important mechanism used by crops to length, but not the number of lateral roots, without any
access more N. effect on primary root growth. In contrast, the application

Ann Appl Biol (2013) 11

2013 Association of Applied Biologists
Do plants need nitrate?
Figure 3 The effect of nitrate supply on lateral root development. (A)
Arabidopsis seedlings were grown for 7 days on 1 mol m3 potassium
nitrate (KNO3 ) compared with those grown on 50 mol m3 KNO3 . (B)
Close-up of a typical stunted lateral root from a seedling grown on
50 mol m3 KNO3 . Reproduced with permission from, Zhang et al. (1999);
copyright 1999, National Academy of Sciences, United States of America.
of high concentrations of NO3 (50 mol m3 ) inhibited
the development of lateral root growth (Fig. 3; Zhang
et al., 1999). Auxin has long been known to play a key
role in the elongation of both primary and lateral roots
(Smith & de Smet, 2012). Zhang et al. (1999) used three
auxin resistant mutants aux1, axr2 and axr4 of Arabidopsis
to test for a possible role for auxin in the NO3 stimulated
increase in lateral root growth. The elongation of lateral
root growth was increased by NO3 in the mutants in the
same way as the WT in aux1 axr2, but not in axr4. The
lack of response of lateral root growth to NO3 by the
axr4 mutant, suggested that there is an overlap between
the auxin and NO3 reaction mechanisms.
Linkohr et al. (2002) carried out similar experiments
to Zhang & Forde (1998) and Zhang et al. (1999), using
Arabidopsis grown on agar plates and also grown on a
soil/sand mixture in order to provide a more realistic
12
M. Andrews et al.
environment. Nitrate stimulated both the length and the
initiation of new lateral roots in the NO3 patch, in
contrast to the findings of Zhang & Forde (1998). Linkohr
et al. (2002) also went on to show that the auxin resistant
mutants axr1, axr4 and aux1 all had wild type responses of
lateral root growth to NO3 patches and concluded that
auxin is not involved in changes in root architecture in
response to NO3 .
The NO3 induced increase in meristematic activity of
the lateral roots, was shown to be a process regulated
by ANR1, a MADS box transcription factor (Zhang &
Forde, 1998; Gan et al., 2012). It was later shown that
NRT1.1 also has a role in this process and acts as a
positive regulator of ANR1 (Remans et al., 2006). NRT1.1
was initially isolated as a NO3 transporter (Wang et al.,
2012b), but there is now evidence that it has a regulatory
role in a large number of NO3 responses. It has recently
been proposed that NRT1.1 is able to act as a transceptor,
a regulator that is known to operate in yeast and animals
(Walch-Liu & Forde, 2008; Ho et al., 2009; Krouk et al.,
2010a; Gojon et al., 2011).
The possible involvement of auxin in the stimulation
of lateral root growth by NO3 was reconsidered, when
it was found that the NO3 transceptor NRT1.1 was
able to transport auxin as well as NO3 (Krouk et al.,
2010b). It was proposed that under low NO3 conditions,
auxin was transported away from the lateral root tip
by NRT1.1 thus suppressing growth. However, following
the application of NO3 , auxin transport by NRT1.1 was
inhibited and thus auxin accumulated and stimulated
growth of the lateral roots (Krouk et al., 2010b; Gojon
et al., 2011). Celis-Aramburo et al. (2011) showed with
Capsicum chinense that NO3 , when applied locally as
a patch, stimulated an increase in both the number
and length of the lateral roots. Auxin could substitute
for NO3 in this stimulation, although differences were
noted between accessions. Ruffel et al. (2011) provided
evidence that cytokinin as well as auxin is involved in
the compensatory increase in lateral root growth in NO3
patches.
The effect of NO3 on primary root growth has been
shown to be less than on lateral roots, but more varied
(Granato & Raper, 1989; Zhang & Forde, 1998; Linkohr
et al., 2002). Walch-Liu & Forde (2008) demonstrated
that NO3 is able to stimulate primary root growth in a
number of Arabidopsis accessions. In contrast, inhibition of
primary root growth by NO3 has been demonstrated in
maize (Zhao et al., 2007; Tian et al., 2008) and NO3 inhib-
ited the growth of the primary root of C. chinense between
the fourth and fifth day of treatment, an effect that could
be reversed by auxin (Celis-Aramburo et al., 2011).
More recently, systems analysis has been used to
investigate how N availability controls root architecture
Ann Appl Biol (2013)
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M. Andrews et al.
(Gutierrez, 2012). In an initial study, Gifford et al. (2008)
showed that the small RNA miR167 and auxin response
factor ARF8 are involved in the NO3 regulation of lateral
root growth in Arabidopsis. Similar small miRNAs were
also shown to interact with auxin response factors in the
regulation of adventitious rooting (Gutierrez et al., 2009).
Later, the small RNA miR393 was shown to be induced by
NO3 in Arabidopsis (Vidal et al., 2010) and to target the
auxin receptor AFB3 in roots. Analysis of afb3 insertional
mutants and miR393 overexpressors demonstrated that
NO3 is able to transcriptionally induce the expression of
AFB3 in both primary and lateral roots, while metabolites
after NO3 reduction were able to downregulate AFB3
due to the induction of miR393. The authors proposed
that miR393/AFB3 is a unique N-responsive feed-forward
mechanism that controls root system architecture in
Arabidopsis (Vidal et al., 2010).
In comparison with NO3 , much less is known about
the effect of NH4 + ions on root architecture. In the early
experiments of Drew (1975), NH4 + exerted a very similar
stimulatory effect to NO3 on lateral root growth in
barley. However, the shape of the lateral roots grown
on NH4 + was described as more tortuous and appeared
denser in the diagrams shown. Lima et al. (2010) using
Arabidopsis growing in sterile culture, confirmed that
NH4 + stimulated highly branched lateral root formation,
while the roots were more dense in NH4 + , the total
increase in root length was less than with NO3 . The
authors proposed that NO3 and NH4 + induced different
responses in root growth and that NH4 + promoted
initiation rather than elongation of lateral roots. The
addition of NH4 + plus NO3 synergistically induced both
the number and length of lateral roots. Lima et al. (2010)
went on to show that the NH4 + transporter AMT 1.3 is
an important regulator of the NH4 + response of lateral
root growth. Rogato et al. (2010a,b) also suggested that in
Lotus japonicus, the AMT 1.3 protein was involved in the
inhibitory growth response to NH4 + . In addition, NH4 +
reduced the growth of the primary roots of Arabidopsis
by inhibiting cell elongation rather than cell division
(Li et al., 2010). When NH4 + was applied to the shoots
of Arabidopsis, there was a reduction in the development
and elongation of lateral roots. It was proposed that lateral
primordium emergence was inhibited by a reduction in
auxin transport from shoot to root by the influx carrier
AUX1 (Li et al., 2011). Very recently, it was shown
that root gravitropism in Arabidopsis is blocked by NH4 +
through auxin redistribution (Zou et al., 2012). In field
experiments in calcareous soils, the localised application
of P with NH4 + , but not NO3 or urea, greatly stimulated
root proliferation in maize (Jing et al., 2010).
Amino acids, when added singly or in groups at high
concentrations, can have an inhibitory effect on plant
Ann Appl Biol (2013)
2013 Association of Applied Biologists
Do plants need nitrate?
Figure 4 The effect of L-glutamate on root architecture of Thlaspi
caerulescens. Seeds were initially germinated under sterile conditions
on a growth medium containing 0.5 mol m3 glutamine as a background
N source. After 6 days of growth, and before lateral roots had appeared,
the seedlings were transferred to a fresh medium containing either
1 mol m3 potassium chloride (control) or 1 mol m3 potassium L-
glutamate (glutamate treated) and incubated for a further 9 days before
image capture. Arrows indicate the locations of the primary root tips at
the time of transfer. Reproduced with permission from Walch-Liu et al.
(2006a); Copyright John Wiley & sons.
growth, due to feedback inhibition of specific biosynthetic
pathways (Bonner & Jensen, 1997). However, when
added singly, only glutamate of the 20 amino acids that
normally occur in protein, had an effect on root growth
of Arabidopsis ecotype C24 (Walch-Liu et al., 2006b).
Glutamate at low concentrations caused inhibition of
primary root growth and stimulation of lateral root
growth immediately behind the root tip, with lateral
roots only themselves becoming sensitive to glutamate
after a developmental delay of several days (Fig. 4). The
precise concentrations of glutamate required to produce
the responses detected at the root tip, varied considerably
between different accessions of Arabidopsis. Roots treated
with related amino acids such as aspartate, GABA or
glutamine did not exhibit any growth inhibitory effects
at the low concentrations that glutamate was effective.
The glutamate inhibitory response was also detected in
the roots of other species of Brassicaceae, Papaveraceae
and Solanaceae (Walch-Liu et al., 2006a,b). At least 20
glutamate-like receptor (GLR) genes have been identified
in plants, which are similar to those found in mammals.
Plants lacking, or overexpressing, the genes have been
characterised and shown to have changes in a wide range
of physiological properties (Forde & Walch-Liu, 2009).
Very recently it was demonstrated that GLRs are able
to function as amino acid gated Ca2+ channels (Michard
et al., 2011; Vincill et al., 2012).
Nitrate was able to prevent the response to glutamate
if it was supplied at the same time to the root tip, but
glutamine and NH4 + had no effect (Walch-Liu & Forde,
13
Do plants need nitrate?
Figure 5 Model for interacting nitrate (NO3 ) and L-glutamate (Glu)
signalling pathways that regulate meristematic activity at the root tip.
Glu sensed at the primary root tip by an unknown receptor triggers a
reduction in primary root growth and increased branching behind the
root tip. NO3 sensed by NRT1.1 at the primary root tip antagonises the
Glu signalling pathway and alleviates the effect of Glu on root architecture.
The model suggests that NRT1.1 is only active as a NO3 sensor when
phosphorylated by a kinase at Thr101 (shown as P on the NRT1.1
protein). It is probable that phosphorylation is reversible and is regulated
by the N supply (Liu & Tsay, 2003). Reproduced with permission from
Forde & Walch-Liu (2009); Copyright John Wiley & sons.
2008; Forde & Walch-Liu, 2009). Evidence obtained
by using mutants of Arabidopsis indicated that NRT1.1
but not ANR1 is an essential component of the NO3
suppression of glutamate effect. NRT1.1 is probably only
able to prevent the glutamate inhibitory effect when it
is phosphorylated by a kinase at a specific threonine
(Thr101) residue on the protein (Fig. 5), a process that is
regulated by the availability of NO3 (Liu & Tsay, 2003).
The data discussed above suggest that of the different N
sources, NO3 is clear cut and results in increased lateral
root growth in most species that have been studied in
detail. However, the interaction of NO3 and auxin in the
control of primary and in particular lateral root growth
is clearly very complex and requires further detailed
studies. The effects of NH4 + are more varied as there is
often growth inhibition, but this may only be when there
is no other N source available and there is no pH buffering
capacity in the soil, which would be unusual. Inhibition
of primary root growth but stimulation of lateral root
growth by glutamate in organic compound rich patches,
in otherwise N poor soil, would allow the plant to
make use of this additional N supply, particularly at
the soil surface and could confer a competitive advantage
(Robinson, 1994; Hodge et al., 1999; Trinder et al., 2012).
Environmental stress/change and N form
Our focus has been on the mechanisms of N form effects
on major plant processes under non-stressed conditions
14
M. Andrews et al.
but it is acknowledged that the magnitude of these effects
is likely to be dependent on environmental variables
outside the supply of N. For example, the uptake of
different N forms can be differentially affected by a change
in temperature. For Lolium multiflorum and L. perenne
on low N supply (0.5 mol m3 NH4 + + 0.5 mol m3
NO3 ), the uptake of NH4 + was greater than NO3 at
root temperatures from 5 to 25 C (Clarkson & Warner,
1979; Clarkson et al., 1986). The uptake of both N
forms decreased with a lowering of root temperature
but for most cultivars tested, this decrease was greater
with NO3 than NH4 + and thus plants exhibited a
greater reliance on NH4 + at low temperatures. However,
the increased selection of NH4 + from an NH4 + /NO3
mix with decreased temperature, does not hold for all
plant species. For two cultivars of barley supplied with
0.0050.2 mol m3 NH4 NO3 in a flowing nutrient culture
system, NH4 + uptake was greater than NO3 at all
concentrations and temperatures from 2.5 to 15 C but
uptake of both N forms was equally inhibited at lower
temperatures (Bloom & Chapin, 1981). Similarly, for
Eucalyptus nitens grown on 0.1 mol m3 NH4 NO3 , uptake
rates of NH4 + and NO3 were both reduced by 70% with
a decrease in root temperature from 20 to 10 C (Garnett
& Smethurst, 1999).
It has already been highlighted that chilling tempera-
ture can interact positively with NO3 in its reduction of
seed dormancy of many species. However, there is strong
evidence that the usually positive effects of increased
NO3 supply on leaf growth can result in leaf damage at
chilling and freezing temperatures (Andrews et al., 1989,
2004). For common bean, growth at chilling temperature
(15 C) was substantially greater at low (1 mol m3 ) than
at high (525 mol m3 ) NO3 supply: the reverse was
found at optimum temperatures (Andrews et al., 1989).
The water content of the central leaflet of the first and
second trifoliate leaves was substantially greater at high
rather than low NO3 supply at 15 C and the expan-
sion rate was initially greater at high NO3 supply but
it was not maintained and the leaf became chlorotic. It
was proposed that the greater rate of leaf expansion asso-
ciated with increased leaf water content, was the cause
of increased leaf damage at higher NO3 supply. Specif-
ically, if the rate of leaf expansion increases, then the
demand for membrane synthesis is greater. Low temper-
ature decreases the rate of incorporation of lipid material
into membranes (Willing & Leopold, 1983) and if the
increased demand for membrane synthesis is not met,
cellular disruption will result. This hypothesis needs test-
ing as chilling affects many plant processes (Vella et al.,
2012). It is likely that increased leaf water content associ-
ated with increased NO3 transport to the shoot, would
make the plants more vulnerable to freezing damage but
Ann Appl Biol (2013)
2013 Association of Applied Biologists
M. Andrews et al.
again this needs testing. Thus, a shift from a root to a
shoot dominant site of NO3 assimilation may increase
low temperature sensitivity, possibly linked to increased
leaf water content (Andrews et al., 2004). Conversely,
and also of agricultural relevance, if common bean and
other species with a shoot dominant site of NO3 assim-
ilation had greater reliance on root NO3 assimilation,
they might be more tolerant of chilling temperatures.
These proposals warrant further study.
There are reports for several species including maize,
wheat, pea, grey poplar (Populus tremula alba) and
Nerium oleander of greater tolerance of moderate salinity
(30100 mol m3 NaCl) following growth under NO3
in comparison with NH4 + nutrition (15.5 mol m3 N)
(Lewis et al., 1989; Speer et al., 1994; Frechilla et al., 2001;
Ehlting et al., 2007; Abdolzadeh et al., 2008). However,
there are also reports for peanut, soybean, wheat and
kaller grass of similar effects of salt on growth on the two
N forms (Silberbush & Lips, 1988; Bourgeais-Chaillou
et al., 1992; Botella et al., 1997; Mahmood & Kaiser,
2003). Reduced growth with NH4 + in comparison with
NO3 under saline conditions is likely to be related to
greater disruption of ionic/nutrient balance (Speer &
Kaiser, 1994; Frechilla et al., 2001). This can occur for
several reasons. For example, there is evidence that pea
has reduced capacity for vacuolar salt sequestration under
NH4 + in comparison with NO3 nutrition (Speer & Kaiser,
1994). Also, Frechilla et al. (2001) reported that reduced
growth of pea under NH4 + rather than NO3 nutrition
was associated with greater total plant Na+ accumulation.
Reduced growth of N. oleander with NH4 + was related to
greater transport to, and accumulation of, Na+ and Cl
in the shoot (Abdolzadeh et al., 2008).
Increased atmospheric CO2 concentration is an envi-
ronmental change which will be experienced by most
plants in the future, and for this reason, the interactions
of CO2 with N assimilation and the growth of plants on
NO3 and NH4 + nutrition have been studied in some
depth. In particular, change in the assimilatory quotient
(AQ) has been used to assess the effect of increased CO2
concentration on NO3 photoreduction/assimilation in a
range of plant species (Bloom et al., 2012). The assimila-
tory quotient (AQ) is the ratio of net CO2 consumption
to net O2 evolution in a photosynthetically active tissue.
This ratio varies between plants assimilating NO3 or
NH4 + because transfer of electrons to NO3 and NO2
during NO3 photoreduction/assimilation increases O2
evolution from the light-dependent reactions of photo-
synthesis with little effect on CO2 fixation in the light
independent reactions. Therefore, plants that are carry-
ing out NO3 photoreduction/assimilation exhibit a lower
AQ than those on NH4 + nutrition and the difference in
the AQ between plants assimilating NO3 or NH4 + is
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Do plants need nitrate?
proportional to NO3 photoreduction/assimilation. In an
initial study on wheat in liquid culture, the AQ was
substantially greater in plants grown on 0.2 mol m3 NO3
than 0.2 mol m3 NH4 + at 360 mol CO2 mol1 but at
700 mol CO2 mol1 the value for NO3 dropped to
close to zero, similar to that for NH4 + (Bloom et al.,
2002). For NO3 fed plants, in vitro shoot NR and
NiR decreased 12 and 27%, respectively on a protein
basis and 33 and 30%, respectively on a fresh weight
basis with increased CO2 concentration from 360 to
700 mol CO2 mol1 . Root NR and NiR were unaf-
fected by CO2 supply. It was concluded that increased
CO2 inhibited shoot NO3 assimilation (Bloom et al.,
2002). Subsequent work determining the AQ indicated
that at 0.2 mol m3 NO3 in liquid culture, an increase
in CO2 concentration from 360400 to 700720 mol
CO2 mol1 caused a decrease in NO3 photoreduction
in seven other C3 species (Arabidopsis, barley, Acer saccha-
rum, Flaveria pringlei, Liquidambar styraciflua, Pinus taeda
and Sequoiadendron giganteum), but had little effect on
three C4 species (maize, Amaranthus retroflexus and Flaveria
bidentis). Two C3 C4 intermediate species (Flaveria chlo-
raefolia and F. sonorensis exhibited intermediate responses
to elevated CO2 (Rachmilevitch et al., 2004; Bloom et al.,
2010, 2012).
Bloom et al. (2010, 2012) outlined three possible
mechanisms to explain the decrease in NO3 photore-
duction/assimilation with increased atmospheric CO2
concentration in C3 but not C4 plants. First, it was argued
that a lower rate of photorespiration caused by elevated
CO2 in C3 plants would result in decreased export of
malate from the chloroplasts and decreased NADH avail-
ability for NO3 reduction in mesophyll cells. In contrast,
increased CO2 has little impact on malate and NADH lev-
els in the cytoplasm of mesophyll cells of C4 plants. The
second mechanism proposed was decreased NO2 influx
into the chloroplast. Nitrite transport from the cytoplasm
into the chloroplast involves the diffusion of HNO2 or
cotransport of H+ and NO2 across the chloroplast mem-
brane which requires the stroma to be more alkaline
than the cytosol. Increased CO2 can dissipate some of this
pH gradient because additional CO2 movement into the
chloroplast acidifies the stroma. Also, enhanced CO2 fixa-
tion hydrolyses ATP faster and requires supplementary H+
exchange across the thylakoid membrane to regenerate
this ATP. The third mechanism relates to increased com-
petition between biochemical processes for Fd reductant
in the chloroplast stroma. The C3 reductive photosyn-
thetic C cycle, the reduction of NO2 to NH4 + and the
assimilation of NH4 + into amino acids all occur in the
chloroplast stroma and all require reduced Fd generated
by photosynthetic electron transport. Elevated CO2 stim-
ulates the Calvin cycle and under light limited conditions,
15
Do plants need nitrate?
can diminish the amount of reduced Fd available for
NO2 reduction or NH4 + assimilation.
Bloom et al. (2012) reported that, at 400 mol CO2
mol1 , Arabidopsis and wheat exhibited greater fresh
weight growth under NO3 , than under NH4 + nutrition,
but A. saccharum, P. taeda and L. styraciflua grew faster with
NH4 + . Under NH4 + nutrition, an increase in atmospheric
CO2 concentration to 720 mol CO2 mol1 , for several
weeks, stimulated growth of Arabidopsis, wheat and P.
taeda but had no effect on the growth of A. saccharum,
and L. styraciflua. Under NO3 nutrition, the increase
in CO2 concentration had no effect on the growth of
Arabidopsis and wheat, but inhibited the growth of the
three tree species. It was concluded that increased CO2
concentration inhibits shoot NO3 assimilation in a wide
variety of C3 plants and this can greatly reduce their
growth. Increased CO2 does not inhibit the growth of
C3 plants on NH4 + nutrition or C4 plants on NH4 + or
NO3 nutrition. Consequently, increasing atmospheric
CO2 concentrations should favour C3 species on sites
where NH4 + is the dominant N source and C4 species
where NO3 is dominant. Thus rising CO2 and its effect on
shoot NO3 assimilation may influence the distribution
of C3 and C4 plants.
Reports for wheat indicate that the effects of increased
CO2 concentration on N uptake, N assimilation and the
growth of plants under NO3 or NH4 + supply may be to
some extent related to the length of exposure to increased
CO2 . For wheat, NO3 depletion from the medium indi-
cated similar rates of uptake of NO3 at 380 and 720 mol
CO2 mol1 but 15 N and 14 N NO3 labelling indicated
that the same increase in CO2 concentration resulted
in decreased NO3 uptake in the short term of 212 h
(Rachmilevitch et al., 2004; Bloom et al., 2010). Bloom
et al. (2002) reported that over a 2 week experiment, the
dry weight of wheat plants grown on 0.2 mol m3 NH4 +
or 0.2 mol m3 NO3 was similar at 360 mol CO2 mol1
but greater with NH4 + than with NO3 at 700 mol CO2
mol1 . Increased CO2 supply increased the shoot and
root growth of plants on NH4 + but did not affect the
shoot growth of plants on NO3 , although these plants
did have larger roots. Shoot and root N concentration
changed little with N form or CO2 supply, thus NH4 +
uptake was greater than NO3 uptake at high CO2 supply
but increased CO2 did not reduce NO3 uptake. Bloom
et al. (2012) reported that over a 5 week period, the
growth of wheat was greater with NO3 than NH4 + at
400 and 720 mol CO2 mol1 and concluded that there
must be an alternative mechanism of NO3 assimilation
at high CO2 , for example, root NO3 assimilation.
It is possible that for a particular NO3 supply, increased
photosynthate availability in the shoots of C3 plants
with an increased CO2 supply, could lead to increased
16
M. Andrews et al.
transport of photosynthate to the roots which could be
used for increased NO3 assimilation in the roots. Thus,
roots could assimilate a greater proportion of the NO3
taken up and hence the shoot would play a lesser role.
There is evidence that root NO3 assimilation can be
limited by supply of reductant. For example for barley,
in vivo NR activity (reliant on tissue NADH) and in vitro
NR activity (NADH supplied at optimum levels) were
similar (15.2 and 12.9 mol NO2 g1 dry weight h1 )
at 0.5 mol m3 applied NO3 but at 5.0 mol m3 NO3 ,
the in vivo assay activity was 2.6 mol NO2 g1 dry
weight h1 in comparison with 15.6 mol NO2 g1 dry
weight h1 for the in vitro assay activity (Andrews et al.,
1992). Also, it is possible that for herbaceous species at
least, substantial NO3 assimilation can occur in shoots
without the requirement of photoreduction of NO3 . For
example, the stem can be a major site of NR activity in a
range of species (Andrews et al., 1984).
In some cases, decreased AQ/NO3 photoreduction
in C3 plants following exposure to increased CO2 was
associated with decreased growth while increased CO2
concentration had no effect or stimulated growth of C3
plants on NH4 + nutrition (Bloom et al., 2012). These
results have potential implications for agricultural systems
as NO3 is the main form of N available to crops in
cultivated soil and many crops assimilate substantial
NO3 in the shoot (Andrews et al., 2004). However,
the evidence indicates that C3 crops can sustain increased
growth under CO2 enrichment especially if they receive
high applications of N (Ainsworth & Long, 2005; de
Graaff et al., 2006; Bloom et al., 2012). Bloom et al. (2012)
proposed that application of N increased the availability of
soil NH4 + and this compensated for lower rates of shoot
NO3 assimilation, but this needs testing. The effects of
increased atmospheric CO2 concentration on root and
shoot NO3 assimilation in crops warrants further study.
Indeed, given that there is evidence that plant water use
efficiency, N-use efficiency, low temperature tolerance
and response to increased atmospheric CO2 concentration
are dependent on whether NO3 is assimilated in the
root or shoot, assessment of the importance of root or
shoot NO3 assimilation under different environmental
conditions has emerged as an important area for further
study.
Conclusions and future work
It is concluded that the form of N available to plants
can affect their time and rate of seed germination,
leaf expansion and function, S : R and root architecture.
Available data indicate that:
Nitrate specific reduction of seed dormancy of
Arabidopsis is due to NO3 induction of the ABA
Ann Appl Biol (2013)
2013 Association of Applied Biologists
M. Andrews et al.
catabolic gene CYP707A2, which encodes ABA
8 -hydroxylase and consequently reduces ABA
in seeds. Nitrate may act on seed dormancy of
Arabidopsis and other species via the production of
NO.
The increased rate of mobilisation of seed reserves
of temperate cereals induced by NO3 is due to
increased water uptake as a result of increased
NO3 uptake and accumulation.
The growth of many plant species is greater with
NO3 than with NH4 + as a sole N source, as high
concentrations of NH4 + can cause NH4 + toxicity.
NH4 + toxicity has several causes and for some
species, including tobacco, occurs at low N supply.
The maximum growth of common bean can be
as great with NO3 , urea, glutamine and allantoic
acid as a N source but is achieved with less N with
NO3 .
Greater leaf area and dry matter per unit N
with NO3 in comparison with other forms of
N, could be related to greater NO3 transport to,
and assimilation in, the shoot leading to greater
osmoticum supply and greater leaf area per unit N.
NO3 can play a role in turgor driven expansion of
guard cells but it is not a pre-requisite for stomatal
function.
Differences in dry matter partitioning between the
shoot and roots of plants grown on different forms
of N nutrition are causally linked with differences
in leaf soluble protein concentration.
Nitrate and auxin interact in the control of lateral
root growth of Arabidopsis.
The magnitude of N form effects on plants is dependent
on environmental variables outside N supply. There
is evidence that plant water use efficiency, NUE,
low temperature tolerance and response to increased
atmospheric CO2 concentration are dependent on
whether NO3 is assimilated in the root or shoot.
Assessment of the importance of root or shoot NO3
assimilation under different environmental conditions
warrants further study.
Acknowledgements
J. A. R. has benefited from discussions with Hans Lambers,
Gus Shaver, Sally Smith and Mark Westoby. The
University of Dundee is a registered Scottish charity, No.
SC015096.
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