Annals of Botany 105: 1141– 1157, 2010

doi:10.1093/aob/mcq028, available online at www.aob.oxfordjournals.org

REVIEW: PART OF A SPECIAL ISSUE ON PLANT NUTRITION

Nitrogen uptake, assimilation and remobilization in plants: challenges

for sustainable and productive agriculture
Céline Masclaux-Daubresse*, Françoise Daniel-Vedele, Julie Dechorgnat, Fabien Chardon, Laure Gaufichon
and Akira Suzuki
Institut Jean-Pierre Bourgin (IJPB) UMR 1318, INRA 78026 Versailles Cedex, France
* For correspondence. E-mail [email protected]

Received: 22 September 2009 Returned for revision: 13 November 2009 Accepted: 17 December 2009 Published electronically: 18 March 2010

† Background Productive agriculture needs a large amount of expensive nitrogenous fertilizers. Improving nitro-
gen use efficiency (NUE) of crop plants is thus of key importance. NUE definitions differ depending on whether
plants are cultivated to produce biomass or grain yields. However, for most plant species, NUE mainly depends
on how plants extract inorganic nitrogen from the soil, assimilate nitrate and ammonium, and recycle organic
nitrogen. Efforts have been made to study the genetic basis as well as the biochemical and enzymatic mechanisms
involved in nitrogen uptake, assimilation, and remobilization in crops and model plants. The detection of the lim-
iting factors that could be manipulated to increase NUE is the major goal of such research.
† Scope An overall examination of the physiological, metabolic, and genetic aspects of nitrogen uptake, assim-
ilation and remobilization is presented in this review. The enzymes and regulatory processes manipulated to
improve NUE components are presented. Results obtained from natural variation and quantitative trait loci
studies are also discussed.
† Conclusions This review presents the complexity of NUE and supports the idea that the integration of the
numerous data coming from transcriptome studies, functional genomics, quantitative genetics, ecophysiology
and soil science into explanatory models of whole-plant behaviour will be promising.

Key words: Nitrogen use efficiency, remobilization, quantitative trait loci, natural variation, nitrate transporter,
ammonium assimilation, glutamine synthetase, leaf senescence, nutrient recycling.

IN T RO DU C T IO N ageing, recycling and remobilization. For crops, NUE has

been defined as the grain yield per unit of nitrogen available
Plants have a fundamental dependence on inorganic nitrogen
from the soil, including nitrogen fertilizer. For Arabidopsis
and 85– 90 million metric tonnes of nitrogenous fertilizers are
and other model plants, a physiological NUE index is
added to the soil worldwide annually (Good et al., 2004).
expressed as the fresh or dry matter produced per nitrogen
Nitrogen is one of the most expensive nutrients to supply and
content in the plant (Good et al., 2004). In both cases, NUE
commercial fertilizers represent the major cost in plant pro-
is the product of nitrogen uptake efficiency (NupE) and nitro-
duction. Furthermore, there is serious concern regarding nitro-
gen utilization efficiency (NUtE), which is the optimal combi-
gen loss in the field, giving rise to soil and water pollution.
nation between nitrogen assimilation efficiency (NAE) and
Incomplete capture and poor conversion of nitrogen fertilizer
nitrogen remobilization efficiency (NRE). Until now, most
also causes global warming through emissions of nitrous
plant cultivars have been selected under non-limiting nitrogen
oxide. Lowering fertilizer input and breeding plants with
conditions. Reducing the excessive input of fertilizers without
better nitrogen use efficiency (NUE) is one of the main goals
affecting productivity raises questions about the adaptive
of research on plant nutrition (Hirel et al., 2007). The ability
responses of such cultivars to low nitrogen availability.
of a plant to capture nitrogen from the soil depends on soil
The biochemical mechanisms involved in N uptake, assim-
type, environment and species. It has been estimated that 50–
ilation and remobilization have been widely studied to identify
70 % of the nitrogen provided to the soil is lost (Hodge et al.,
the bottlenecks associated with NUE. Quantitative genetics is
2000). Therefore, improving NUE is essential in order to
also conducted using both crops and model plants to evaluate
reduce damage due to nitrate leaching, ecosystem saturation
the genetic basis of NUE. An overall examination of the phys-
and water pollution. NUE in plants is complex and depends
iological, metabolic and genetic aspects of NUE is presented
on nitrogen availability in the soil and on how plants use nitro-
in this review.
gen throughout their life span. As a concept, NUE is expressed
as a ratio of output (total plant N, grain N, biomass yield, grain
yield) and input (total N, soil N or N-fertilizer applied).
Increasing NUE and limiting nitrogen fertilizer use are both NI TROG EN SO UR CES A ND U PTA KE
important and challenges to preserve the environment and Although generally low, soil nitrogen availability can fluctuate
improve a sustainable and productive agriculture. greatly in both space and time due to factors such as precipi-
The use of nitrogen by plants involves several steps, includ- tation, temperature, wind, soil type and pH. Therefore, the pre-
ing uptake, assimilation, translocation and, when the plant is ferred form in which N is taken up depends on plant adaptation
# The Author 2010. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: [email protected]
1142 Masclaux-Daubresse et al. — Nitrogen in agricultural plants

to soil conditions. Generally, plants adapted to low pH and heterologous systems, to transport not only nitrate but also
reducing soils as found in mature forests or arctic tundra di- or tripeptides, while OPT (oligo-peptide transporter) pro-
tend to take up ammonium or amino acids, whereas plants teins transport tetra/pentapeptides.
adapted to higher pH and more aerobic soils prefer nitrate The high-affinity transport system (HATS), acting when the
(for a review see Maathuis, 2009). external nitrate concentration is low, relies on the activity of
Nitrate uptake occurs at the root level and two nitrate trans- the so-called NRT2 family (reviewed in Williams and
port systems have been shown to coexist in plants and to act Miller, 2001). Although the functional characterization of
co-ordinately to take up nitrate from the soil solution and almost all higher plant NRT2 transporters remains to be
distribute it within the whole plant (Fig. 1) (review in done, it is now well documented that AtNRT2.1, in interaction
Daniel-Vedele et al., 1998; Tsay et al., 2007). with an NAR2 protein (Orsel et al., 2006), is a major com-
It is generally assumed that the NRT1 gene family mediates ponent of the HATS in Arabidopsis, as shown by the fact
the root low-affinity transport system (LATS), with the excep- that a mutant disrupted for the AtNRT2.1 gene has lost up to
tion of the AtNRT1.1, which is both a dual affinity transporter 75 % of the high-affinity NO2 3 uptake activity and showed a
(Wang et al., 1998; Liu et al., 1999) and a nitrate sensor (Ho lower leaf nitrate content (Filleur et al., 2001). As a conse-
et al., 2009). In Arabidopsis, 53 genes belong to the NRT1 quence, growth of these mutants is severely impaired at low
family. Among them 51 genes are expressed and exhibit differ- NO2 3 concentration (Orsel et al., 2004, 2006). A lower
ent tissue expression patterns in the whole plant (Tsay et al., nitrate content was also found in a clce mutant, affected in a
2007), suggesting a specialized and unique function for at protein belonging to the Arabidopsis AtCLC (ChLoride
least some of them. AtNRT1.1 (formerly Chl1) is the most Channel) family. De Angeli et al. (2006) have demonstrated
extensively studied gene and was the first to have been isolated that another member of the family, the vacuolar AtCLCa
(Tsay et al., 1993). The protein is located on the plasma mem- protein, behaves as a NO2 3 /H
þ
exchanger allowing the
brane and the gene is expressed in the epidermis of the root accumulation of nitrate within the vacuole. Insertion mutants
tips and in the cortex and endodermis in the more mature within the AtCLCa gene exhibit normal development but
part of the root (Huang et al., 1999). AtNRT1.2 is constitu- show a reduced capacity to store nitrate but not other anions.
tively expressed only in the root epidermis and participates This phenotype was also recently found when the expression
in the constitutive low-affinity system (Huang et al., 1999). of AtNRT2.7 was affected. This AtNRT2 gene is expressed in
Once taken up by root cells, nitrate must be transported aerial organs and also highly induced in dried seeds. In two
across several cell membranes and distributed in various allelic atnrt2.7 mutants, less nitrate is accumulated in the
tissues. AtNRT1.5, located on the plasma membrane of root seed. In contrast, seeds from plants over-expressing the
pericycle cells close to the xylem, is involved in long-distance AtNRT2.7 coding region accumulate more nitrate and as a con-
transport of nitrate from the root to the shoot (Lin et al., 2008). sequence are less dormant than the corresponding wild-type
The AtNRT1.4 gene is only expressed in the leaf petiole, and in seeds (Chopin et al., 2007).
the mutant the level of nitrate content in the petiole is half that Electrophysiological studies together with the pH-dependent
in the wild-type (Chiu et al., 2004). The AtNRT1.6 gene, equilibrium between the uncharged NH3 and charged NHþ 4
expressed in the vascular tissue of the silique and funiculus, forms suggest that the ion is predominant under all physiologi-
is thought to deliver nitrate from maternal tissue to the devel- cal conditions and is the dominant species for controlled mem-
oping embryo (Almagro et al., 2008). A striking property of brane transport (Ludewig et al., 2007). With functional
the NRT1 family is that certain members belonging to the complementation of a yeast mutant defective in methylammo-
group II (reviewed in Tsay et al., 2007) are able, in nium uptake and recent efforts in sequencing the genome of
model species, 6 genes belonging to the same family of
ammonium transporters were found in Arabidopsis
(Gazzarini et al., 1999), 10 in rice (Sonoda et al., 2003) – a
NRT2.7 species adapted to ammonium nutrition (Yoshida, 1981) –
and 14 in poplar (Couturier et al., 2007). In order to analyse
NRT1.6 the function of each of the AMT (ammonium transporter)
genes separately in planta, physiological and ammonium
influx studies were carried out on single, double, triple and
NRT1.4 quadruple mutants, obtained by T-DNA insertion or by
AMT1;2
RNAi approach, or by complementing the quadruple mutant
AMT1;1
by single genes (Yuan et al., 2007). An additive contribution
AMT1;3 of each protein to ammonium transport was shown, AMT1.1
NRT1.2 and AMT1.3 conferring a similar capacity of 30– 35 % while
NRT2.1 NRT1.5 AMT1;5
NRT2.2
AMT1.2 conferred a lower capacity of 18– 25 %. A second
NAR2 NRT1.1 saturable transport system with a low Km of 4.5 mM and a
very low capacity is thought to be coded by the AMT1.5
F I G . 1. Schematic presentation of the known localisation of NRT1, NRT2 and gene. A complex picture is now emerging from these
AMT genes in Arabidopsis. Two nitrate transport systems have been shown to studies. There is a spatial organization of AMT1 proteins
coexist in plants and to act co-ordinately to take up nitrate from the soil sol-
ution and distribute nitrate within the whole plant. The role of each ammonium
(Fig. 1), the transporters possessing the highest ammonium
transporter has been shown by the study of single, double, triple and quadruple affinities being located in outer root cells or root hairs where
mutants. they can take up ammonium from the soil solution (AMT1.1,
 Masclaux-Daubresse et al. — Nitrogen in agricultural plants 1143

AMT1.3, AMT1.5). The lower affinity of AMT1.1, and its After nitrate reduction, nitrite is translocated to the chloro-
location in the endodermis along the root hair zone, suggests plast where it is reduced to ammonium by the second
a function in the retrieval of ammonium that is released enzyme of the pathway, the nitrite reductase (NiR). The Nii
from the cortex or that enters the root via the apoplastic genes encoding the NiR enzyme have been cloned from
route. The electrochemical gradient between the vacuole and various species, the number of genes varying from one to
cytosol would drive NH3 import to and NHþ 4 export out of two copies (Meyer and Stitt, 2001).
the vacuole. Indeed, tonoplast intrinsic proteins from the TIP Ammonium, originating from nitrate reduction, and also from
family were shown to play a role in NH3 transport into the photorespiration or amino acid recycling, is mainly assimilated
vacuole (Loque et al., 2005). Vacuolar loading with NHþ 4 in the plastid/chloroplast by the so-called GS/GOGAT cycle
should require an electrogenic ammonium transporter, which (Lea and Miflin, 1974; Lea and Forde, 1994). The glutamine
is not yet identified. synthetase (GS) fixes ammonium on a glutamate molecule to
Thus far, putative plant amino acid transporters have been form glutamine. This glutamine reacts subsequently with 2-
identified as members of at least five gene families. In oxoglutarate to form two molecules of glutamate, this step
Arabidopsis these comprise at least 67 genes (reviewed in being catalysed by the glutamine 2-oxoglutarate amino transfer-
Rentsch et al., 2007). Substrate specificities as well as gene ase (or glutamate synthase, GOGAT). The decameric structure
expression patterns or subcellular localization of the protein of maize GS was described by Unno et al. (2006). Two
may give a good indication of the function of each protein. classes of nuclear genes code for GS: the GLN2 and GLN1
Forward and reverse genetic approaches were used to identify genes. GLN2, present as a single nuclear gene in all the
transporters involved in root amino acid uptake (Hirner et al., species studied so far, codes for the chloroplastic GS2,
2006; Svennerstam et al., 2007). Both studies led to the con- thought to be involved in the primary assimilation of
clusion that LHT1 (lysine/histidine transporter), belonging to ammonium coming from nitrate reduction in both C3 and C4
the ATF (amino acid transport) family, is crucial for root plants and in the re-assimilation of ammonium produced from
uptake of acidic and neutral amino acids. The AAP1 (amino photorespiration in C3 plants. The magnitude of the ammonium
acid permease 1) protein was also shown to transport flux through the photorespiration pathway in the leaves of C3
uncharged amino acids but only when they are supplied at plants was indeed estimated to exceed that produced from
high concentrations in the external medium (Lee et al., nitrate reduction by five- to ten-fold (Keys et al., 1978).
2007). Uptake of cationic amino acids such as L-lysine or Conversely, the GLN1 gene family codes for cytosolic GS1
L-arginine is mediated by AAP5 within the concentration isoforms, present in different organs such as roots or stems and
range relevant for field conditions (Svennerstam et al., thought to be involved in ammonium recycling during particu-
2008). The precise localization of these transporter mRNAs lar developmental steps such as leaf senescence and in gluta-
within different cell types in the root led Näsholm et al. mine synthesis for transport into the phloem sap (reviewed
(2009) to propose a hypothetic mode of root amino acid by Bernard and Habash, 2009). Two different forms of gluta-
uptake in non-mycorrhizal plants. mate synthase are present in plants: Fd-GOGAT and
NADH-GOGAT use ferredoxin and NADH as the electron
donors, respectively (Vanoni et al., 2005). Fd-GOGAT is pre-
N I T RO G E N A S S I M I L AT IO N : C RO S S - TAL K
dominantly localized in leaf chloroplasts whereas NADH-
W I T H CA R B O N M E TAB O L I S M
GOGAT is primarily located in plastids of non-photosynthetic
The nitrogen sources taken up by higher plants are nitrate or tissues, such as roots, etiolated leaf tissues and companion
ammonium as inorganic nitrogen sources and amino acids cells. The structural, mechanistic and regulatory properties of
under particular conditions of soil composition. Nitrogen GOGAT enzymes and their role in amino acid metabolism
assimilation requires the reduction of nitrate to ammonium, have been reviewed by Suzuki and Knaff (2005).
followed by ammonium assimilation into amino acids In addition to the GS/GOGAT cycle, three enzymes prob-
(Fig. 2A). ably participate in ammonium assimilation. Cytosolic aspara-
Nitrate reduction takes place in both roots and shoots but is gine synthetase (AS) catalyses the ATP-dependent transfer of
spatially separated between the cytoplasm where the reduction the amido group of glutamine to a molecule of aspartate to
takes place and plastids/chloroplasts where nitrite reduction generate glutamate and asparagine (Fig. 2A; Lam et al.,
occurs. Nitrate reduction into nitrite is catalysed in the 2003). Masclaux-Daubresse et al. (2006) provided evidence
cytosol by the enzyme nitrate reductase (NR) (Meyer and that AS can also use ammonia as a substrate. Indeed, providing
15
Stitt, 2001). This enzyme is a homodimer, each monomer NHþ 4 to leaf discs in the presence of azaserine, the authors
being associated with three prosthetic groups: flavin adenine blocked glutamate biosynthesis but not asparagine biosyn-
dinucleotide (FAD), a haem and a molybdenum cofactor thesis, thus showing that asparagine production is not amino
(MoCo). Characterization of mutants resistant to chlorate, transferase dependent. In Arabidopsis, three genes encode
which can be reduced into toxic chlorite by NR, identified AS (ASN1, ASN2 and ASN3). Asparagine has a higher N/C
two classes of genes, the Nia genes encoding the NR apoen- ratio than glutamine and can be used as a long-range transport
zyme and the Cnx genes encoding the MoCo cofactor. Since and storage compound, especially in legumes (Rochat and
1993, considerable work has been done to characterize the Boutin 1991; Lam et al., 2003). AS could in certain situations
NR in different species (reviewed by Meyer and Stitt, 2001). compensate for the reduced GS-dependent ammonium assim-
Although the NR enzyme is thought to be localized in the ilatory activity.
cytosol, an association with the plasma membrane (PM-NR) Carbamoylphosphate synthase (CPSase) forms carbamoyl-
has been found in corn roots and barley (Ward et al., 1989). phosphate, a precursor of citrulline and arginine, within
1144 Masclaux-Daubresse et al. — Nitrogen in agricultural plants

A
Cytosol

glutamate
glutamate
aspartate
GOGAT

asparagine asparagine glutamate

glutamine
AS AS

glutamine GS2
glutamine
or NH4+ NiR NH4
+

–
GS1 NO2 CPSase
translocation
Phloem sap

carbamoylphosphate
NO2–

NR arginine (storage)

– +
Plastid
NO3 NH4

B
GLU GLN ASN

Phloem sap

Chloroplastid

Dismantling
Respiration

Vacuole
SAV
Proteolysis
Mitochondrion

glutamine
Amino acids
glutamate +
NH4

GS1 GDH

glutamate +
NH4

GS1
+ +
NH4 NH4

N remobilization
asparagine GLN
Cytosol
AS

F I G . 2. Schematic presentation of key enzymes involved in nitrogen management in (A) young and (B) senescing leaves. (A) Nitrate reductase (NR) and aspar-
agine synthetase (AS) are localized in the cytosol, and nitrite reductase (NiR), glutamine synthetase 2 isoenzyme (GS2), glutamate synthase (GOGAT) and car-
bamoylphosphate synthetase (CPSase) within the plastids of mesophyll cells. Glutamine synthetase isoenzyme 1 (GS1) and AS are located in the cytosol of
companion cells. (B) Senescence-associated events include chloroplast degradation and translocation of plastid proteins to the central vacuole via
senescence-associated vacuole (SAV) trafficking. Amino acid recycling occurred in mitochondria and cytosol of mesophyll cells and companion cells.
Glutamate dehydrogenase (GDH), GS1 and AS are the major enzymes involved in the synthesis of glutamine, glutamate and asparagine in the phloem.

plastids using bicarbonate, ATP and ammonium or the amide for GDH in plant cells has been reported to be glutamate dea-
group of glutamine. In Arabidopsis, a single copy each of mination (Masclaux-Daubresse et al., 2006; Purnell and
carA and of carB encode the small subunit and the large Botella, 2007). GDH activity was shown to be essential for
subunit of CPSase, respectively, to form a single heterodimeric plant survival in dark conditions (Miyashita and Good, 2008).
enzyme (Potel et al., 2009). NR, NiR and GOGAT require reducing power as either
Finally, the mitochondrial NADH-glutamate dehydrogenase NADH or ferredoxin (Fdx) according to the enzyme.
could alternatively incorporate ammonium into glutamate in Glutamine synthetase and asparagine synthetase need ATP.
response to high levels of ammonium under stress In addition, carbon skeletons and especially keto-acids are
(Skopelitis et al., 2006). However, the major catalytic activity essential to form organic nitrogen as amino acids. The
 Masclaux-Daubresse et al. — Nitrogen in agricultural plants 1145

availability of carbon skeletons for ammonium condensation shown that leaf senescence is also important for yield.
and the supply of ATP, Fdx and NADH as products of photo- Delaying leaf senescence results in a prolongation of photo-
synthesis, respiration and photorespiration pathways are thus synthesis that increases grain yield and carbon filling into
essential for nitrogen assimilation. seeds. Breeding plants have then to cope with the dilemma
that delayed senescence could lead to higher yields but also
to a decrease in NRE and to a decrease in grain protein
N I T RO G E N R E M O B I L I Z AT I O N : A K E Y FACTOR content. On the other hand, increasing nitrogen remobilization
FO R NI T ROG E N U S E E F F I C I E NC Y has the advantage of re-using nitrogen from the vegetative
parts and of lowering nitrogen loss in the dry remains.
Nitrogen remobilization and yield dilemma
Leaf proteins and in particular photosynthetic proteins of plas-
Nitrogen sources for N remobilization
tids are extensively degraded during senescence, providing an
enormous source of nitrogen that plants can tap to supplement Chloroplasts are the main source of nutrients used during
the nutrition of growing organs such as new leaves and seeds. senescence. Rubisco accounts for 50 % of the total soluble
Nitrogen remobilization has been studied in several plant protein content in the leaves of C3 plants and 20 % in C4
species through the ‘apparent remobilization’ method, which plants (Mae et al., 1983; Sage et al., 1987). Together with
is the determination of the amount of total nitrogen present other photosynthesis-related proteins, Rubisco is a major
in the different plant organs at different times of development source of nitrogen for remobilization. Over-investment in
and through 15N long-term labelling, which allows the deter- Rubisco is thus important for N-source management at the
mination of fluxes (Gallais et al., 2006). In Arabidopsis and whole-plant level.
oilseed rape, it has been shown that nitrogen can be remobi- Although chloroplasts show the first symptoms of deterio-
lized from senescing leaves to expanding leaves at the vegeta- ration during senescence, whereas other organelles are
tive stage as well as from senescing leaves to seeds at the degraded later, the mechanisms responsible for chloroplast
reproductive stage (Malagoli et al., 2005; Diaz et al., 2008; degradation are largely unknown. Chloroplast dismantling
Lemaı̂tre et al., 2008). In Arabidopsis, for which sequential does not mean chaotic decay. Controlled and coordinated
(at vegetative stage) and monocarpic (after flowering) senes- degradation is needed to prevent cell damage due to the
cence phases can be distinguished easily, it was shown that highly photodynamic nature of some of the breakdown pro-
the N remobilization rate was correlated with leaf-senescence ducts and to maintain export capacity and remobilization.
severity at the vegetative stage only (Diaz et al., 2008). The initial steps of chlorophyll and chloroplast protein degra-
Experiments of 15N tracing at the reproductive stage showed dation are likely to take place within the plastid itself (review
that the rate of nitrogen remobilization from the rosettes by Martinez et al., 2008). Later steps may take place within the
to the flowering organs and to the seeds was similar in central vacuole.
early- and late-senescing lines (Diaz et al., 2008). At the Degradation of chloroplast proteins within the organelle is
reproductive stage, NRE is mainly related to harvest index supported by the fact that chloroplasts contain a rather high
(C. Masclaux-Daubresse, unpubl. res.). number of proteases, some of them being upregulated during
Studies using 15N tracing in cereals, oilseed rape and senescence. Chloroplastic DegP and FstH proteases seem to
legumes showed that the onset of grain filling was a critical be responsible for D1 protein degradation during senescence.
phase because N uptake and N2 fixation declined during The FstH6 protease may be implicated in LHCII degradation
plant maturation and seed filling (Salon et al., 2001). (for a review see Martinez et al., 2008). The exact processes
Nitrogen fluxes and 15N remobilization experiments performed leading to Rubisco degradation remain unclear. It was
by Cliquet et al. (1990) showed that leaf blades, stalks, cob, shown that plastids isolated from senescing leaves can
husk and shank serve successively as N-sinks and N-sources. degrade photosynthetic proteins to some extent, particularly
Nitrogen uptake and assimilation during the grain filling in the light (Feller et al., 2008). Proteolysis might be initiated
period is generally insufficient for the high demand of the by the deleterious effects of reactive oxygen species (ROS)
seeds, and the progressive and numerous remobilization over-produced within the organelle during senescence
steps, occurring successively in the different plant organs, (Zimmermann and Zentgraf, 2005). Toxic effects of ROS are
are needed to route nitrogen to the seeds. The contribution usually balanced by a battery of anti-oxidative enzymes.
of leaf N remobilization to rice, wheat or maize grain N During leaf senescence, this equilibrium is altered and ROS
content is cultivar dependent, varying from 50 to 90 % over-production may occur. The degradation of stromal pro-
(Masclaux et al., 2001). N remobilization is also environment teins such as Rubisco and chloroplastic glutamine synthetase
dependent and favoured under limiting nitrate supplies (GS2) generated via the action of ROS was shown to release
(Lemaı̂tre et al., 2008). Although 15N remobilization is a 37- and 16-kDa and 39- and 31-kDa degradation products,
step-by-step mechanism that involves the different plant respectively (Ishida et al., 1997; Palatnik et al., 1999).
organs, evidence shows that grain nitrogen content is corre- The senescence-induced aspartic protease CND41 encoded
lated with flag leaf senescence in maize, wheat and barley. by the nuclear genome has been localized specifically to the
Flag leaf senescence seems then to have a special role in nitro- chloroplast and an in vitro analysis confirmed that CND41
gen availability for grain filling. The onset and the speed of showed a Rubisco proteolytic activity at physiological pH
flag leaf senescence are likely to be essential for NRE (Kato et al., 2005). However, data suggest that CND41 could
(Martin et al., 2005; Uauy et al., 2006). Leaf senescence is not act on Rubisco unless its structure was denatured.
not only essential for nitrogen mobilization. Evidence has Therefore, active Rubisco in the chloroplast would be resistant
1146 Masclaux-Daubresse et al. — Nitrogen in agricultural plants

to CND41-catalysed degradation until leaf senescence was vesicle. Because SAG12 is the only SAG whose expression
under way. CND41 homologues identified in Arabidopsis is uniquely controlled by natural senescence, a specific role
accumulate with ageing. Their role in the regulation of of SAV in the natural senescence process has been proposed.
Rubisco turnover and senescence in Arabidopsis remains to Regulation of proteolysis during senescence is then likely to
be studied (Kato et al., 2005; Diaz et al., 2008). integrate the regulation of chloroplastic and vacuolar proteases
It seems likely that degradation of chloroplast protein starts and the regulation of various trafficking pathways. Desclos
within the plastid but it is far less clear whether chloroplastic et al. (2009) showed that during the nitrogen remobilization
proteases drive protein degradation to the end. The central phase occurring in oilseed rape leaf senescence, the downregu-
vacuole that remains intact and for which compartmentation lation of a protease inhibitor named BnD22 parallels the
is maintained during senescence might be the end point of increase of numerous proteases, including SAG12. The pro-
chloroplast protein degradation. The increase of vacuolar pro- tease activation state might thus be tightly controlled during
tease mRNA levels in senescing leaves is in good agreement leaf development in relation with N remobilization (Etienne
with this scenario (Martinez et al., 2008). Several possible et al., 2007; Desclos et al., 2009).
routes for internalization of chloroplast components by the
central vacuole are proposed, such as autophagosome and
Plant nitrogen uptake and metabolism during senescence
senescence-associated vesicle (SAV) trafficking.
Autophagy is a well-known process in yeast and animals Depending on the species, nitrogen uptake could be nega-
but it has only been recently established in plants. Macro- tively regulated or even in some cases totally inhibited
autophagy involves formation of autophagosomes, which are during seed production. For example, in oilseed rape, the tran-
double membrane-bound structures enclosing macromolecules sition between the vegetative and reproductive phases is
and organelle residues. Transcriptome analysis has shown that characterized by a drastic decrease of HATS and HATS þ
most of the autophagy genes are upregulated during senes- LATS activities (Malagoli et al., 2004). This change is corre-
cence and in response to nitrogen limitation (Thompson and lated with a strong repression of gene expression, the BnNRT2
Vierstra, 2005; Wingler et al., 2009). Chiba et al. (2003) mRNA being undetectable at the flowering stage (Beuve et al.,
demonstrated that Rubisco is released from the chloroplast 2004). In contrast, in maize, post-silking nitrogen uptake con-
into Rubisco-containing bodies (RCBs) in naturally senescent tributes 30 – 70 % of the total N in the seeds, according to the
leaves. Concanamycin A is an inhibitor of vacuolar environment and the genotype (Coque and Gallais, 2007). In
Hþ-ATPase activity that blocks autophagosome degradation. Arabidopsis, when plants are grown under non-limiting N
When leaves of transgenic Arabidopsis plants expressing supply (10 mM nitrate), nitrate influx (HATS þ LATS) is
stromal-targeted fluorescent proteins were incubated with con- lower in the reproductive than in the vegetative stage but
canamycin A, spherical bodies exhibiting protein-specific flu- still operates during seed maturation (Fig. 3). This residual
orescence were observed within the vacuolar lumen. These influx is correlated with a decreased yet significant expression
bodies – corresponding to RCBs – were, however, not of both NRT2.1 and NRT1.1 genes (Fig. 3B; J. Dechorgnat
observed in the concanamycin A-treated leaves of the atg5 et al., unpubl. res.). However, due to this decrease in N
T-DNA insertion mutant impaired for autophagy. In addition, uptake activities, another nitrogen source such as remobiliza-
stromal-targeted DsRed proteins and GFP-ATG8 fusion pro- tion is necessary to cope with the strong N demand from
teins were observed together in autophagic bodies within the seed filling.
vacuole. The authors concluded that Rubisco and stromal pro- There is evidence that plants share common N remobiliza-
teins can be mobilized to the vacuole through an ATG gene- tion mechanisms whether they are monocotyledonous, dicoty-
dependent autophagic process without prior chloroplast ledonous, C3 or C4 photosynthesis types. Grain N
destruction (Ishida and Yoshimoto, 2008). The role of autop- accumulation usually appears to be regulated by the N
hagy in recycling cell proteins is now accepted, although the source. In wheat, the kinetics of Rubisco content and grain
premature leaf senescence and accelerated chloroplast degra- N accumulation suggest that during grain filling N transloca-
dation observed in autophagy knockout lines is less well tion from the vegetative organs is mainly limited by the avail-
understood. As shown by yeast and animal studies, autophagy ability of the substrate in the source organs (Bertheloot et al.,
might have a dual role preventing or triggering cell death 2008, and references therein). To investigate whether NRE is
depending on its fine-tuning. By removing cell waste, autop- controlled by source availability or by the transfer processes
hagy could be essential for cell longevity. In contrast, exces- located in the source leaves and the phloem pathway effi-
sive autophagy activity could lead to cell death. ciency, functional genomic and mutant approaches have been
SAVs differ from autophagosomes in that they occur only in used.
chloroplast-containing cells whereas autophagosomes have Genes encoding enzymes involved in nitrogen metabolism
been observed in leaf and root cells (Otegui et al., 2005; and specifically induced during N remobilization have been
Xiong et al., 2005). As with RCBs, evidence has shown that identified (Masclaux et al., 2001; Buchanan-Wollaston et al.,
SAVs contain chloroplast proteins such as Rubisco and GS2 2003; Guo et al., 2004). These enzymes are a major focus of
(Martinez et al., 2008). Because of their high protease activity, plant physiologists, with special focus on cytosolic glutamine
SAVs are a likely site for chloroplast protein degradation. The synthetase (GS1), glutamate dehydrogenase (GDH) and AS
senescence-associated cysteine-protease encoded by the (reviewed by Masclaux-Daubresse et al., 2008). Nitrogen
SAG12 (senescence-associated gene) gene has been detected assimilation and recycling in young leaves mainly takes
in SAVs (Otegui et al., 2005). SAG12 may then participate place within the chloroplast where nitrite reduction occurs
in the intense proteolytic activity contained in this type of and ammonium is assimilated by the GS/GOGAT cycle
 Masclaux-Daubresse et al. — Nitrogen in agricultural plants 1147

A B NRT2-1

Nitrate influx (µmol 15N h–1 g–1 of root d. wt)

1200
30
1000

Expression (% TUB4)
25

800
20

600
15
NRT1-1
10 400

5 200

0 0
Veg. Repro. Veg. Repro. Veg. Repro.

F I G . 3. Nitrate uptake (HATS þ LATS) is lower during seed maturation than in the vegetative stage but still operates. (A) Root nitrate influx in plants at the
vegetative (Veg.) and reproductive (Repro.) stage. Nitrate influx was measured by supplying 6 mM 15NO2 3 for 5 min to plants grown with 10 mM nitrate. (B)
Expression of NRT1-1 (left) and NRT2-1 (right) genes, at the vegetative (Veg.) and reproductive (Repro.) stage. Expression of nitrate transporter genes was
measured using quantitative PCR and expressed as a percentage of the tubulin 4 gene, used as a control.

involving chloroplastic GS2 and Fd-GOGAT (Fig. 2A). cells and that the mutant plants accumulate amides in their
Chloroplast breakdown during senescence involves de facto old leaves. However, 15N labelling experiments did not show
NiR, GS2 and GOGAT proteolysis. In senescing leaves, nitro- significant differences between mutant and wild-type for
gen recycling and re-assimilation needs then to be catalysed by N-remobilization (J. Lothier et al., INRA, Versailles, France,
enzymes other than chloroplastic ones. The metabolic model unpubl. res.).
proposes that glutamine is mainly synthesized in senescing Among the five GS1-encoding genes (Gln1-1 to Gln1-5) in
leaves by newly expressed GS1 isoforms (Fig. 2B). Using maize, only Gln1-4 is upregulated during senescence (Martin
the amino acid pool released via the proteolysis of chloroplast et al., 2005, 2006). Gln1-3 and Gln1-4 knockout mutants
proteins, a series of transamination reactions would lead to an have been isolated (Martin et al., 2006). The gln1-3, gln1-4
increase in the glutamate pool that could serve immediately as and gln1-3.gln1-4 double mutants showed a sharp reduction
a substrate for GDH, deaminating activity thus providing 2- of kernel yield whereas nitrogen concentration in the kernels
oxoglutarate and ammonia. Ammonia released this way is in was increased. The gln1-3 and gln1-4.gln1-3 mutants accumu-
turn re-assimilated by GS1 to produce glutamine for export. lated large amount of amino acids and ammonia in the source
The importance of GS1 in nitrogen management, growth leaf located below the ear and dedicated to grain feeding.
rate, yield and grain filling has been emphasized by functional Amino acid accumulation in the blade was mainly due to an
genomics and quantitative trait loci (QTL) approaches mainly increase in glutamate and asparagine levels as a consequence
performed on rice and maize (Hirel et al., 2001; Obara et al., of a dysfunction in N export that reduced the total amino
2004; for a review see Bernard and Habash, 2009). Correlation acid concentration and especially glutamine amounts in the
between GS activity and the amount of N remobilized from phloem sap of mutants. Interestingly, the Gln1-4 locus
shoot to the grain was demonstrated in wheat using cultivars co-localized with a maize QTL for thousand-kernel weight,
exhibiting contrasted NUE (Kichey et al., 2007) and using a and the Gln1-3 locus co-localized with two QTLs for
quantitative genetic approach (Habash et al., 2007). thousand-kernel weight and yield. More recently, 15N tracing
However, the role of the GS1 enzyme is complex – numerous experiments showed that the Gln1-2 and Gln1-4 loci
isoforms encoded by a multigenic family exist. In rice, three co-localized with a QTL for N remobilization (Hirel et al.,
genes have been identified, while maize and Arabidopsis 2001; Coque et al., 2008). The role of Gln1-2 remains to be
contain five GLN1 genes coding for GS1 (Bernard and determined.
Habash, 2009). These genes are not regulated in a similar In rice, mutants lacking OsGS1.1 were severely impaired in
manner and GS1 isoforms are located in different plant growth rate and grain filling. Total free amino acid concentration
tissues and do not have the same kinetic properties was reduced in leaf blades of this mutant due to low glutamine
(Ishiyama et al., 2004). It is thus clear that not all GS1 iso- levels. The OsGS1.1 gene product, which is located in compa-
forms participate equally in N remobilization. nion cells and parenchyma cells of leaf tissues, is likely to be
In Arabidopsis, transcriptomic data show that all GLN1 responsible for the generation of glutamine for remobilization
genes except GLN1.5 are induced by leaf senescence (Guo via the phloem (Obara et al., 2001, 2004).
et al., 2004). GLN1.1 was induced more than five-fold. Efforts to study nitrogen management during leaf senes-
Functional genomics are in progress to determine the extent cence have mainly focused on GS1 and to a lesser extent on
of the participation of each of the four senescence-induced GDH. However, much data support the idea that GS1 and
GLN1 genes in N remobilization. Our recent results on GDH are not the only limiting factors in N remobilization
gln1.2 mutants show that GLN1.2 is expressed in companion (for a review see Masclaux et al., 2001). Transcriptomic
1148 Masclaux-Daubresse et al. — Nitrogen in agricultural plants

A B

T1
T0 T1 15N-labelled plants
New leaves
15N
Rosette Rosette
Roots Roots
Chase period
Col0 asn1 Col0 asn1
2 weeks 14N

Labelling period
5 weeks 15N
Day 0 Col0
Chase period
120 14N asn1

partitioning in shoots
T0

(as % of whole-plant)
C 100 T1
0·7
2·5
0·6 80
WT 2·0
0·5 M 60
0·4 1·5 40
0·3 20
1·0
15N

0·2
0
0·5 Rosette Rosette New leaves
0·1
(same leaves (T0–T1)
0 0 as T0)
Harvest index (HI) P% 15N P% 15N/HI

F I G . 4. Asparagine synthetase AS1 might play a role in nitrogen recycling and mobilization. (A) Phenotypes of asn1 mutant (Gabi 829B05) and Col0 wild-type
grown in greenhouse with 10 mM nitrate. (B) Hydroponically grown asn1 mutant (Gabi 829B05) and Col0 were labelled using 15NO2 3 -containing nutritive sol-
ution for 4 weeks (from sowing to the end of the pulse period T0). At T0, plants were transferred to a 15NO23 -free solution and chase period was performed over 2
weeks (T1 ¼ T0 þ 2 weeks). At T1, partitioning of 15N (15N%, as percentage of whole plant) in roots, rosette (already emerged at T0) and new leaves (emerged
between T0 and T1) was monitored in ans1 mutant and Col0. Nitrogen remobilization from rosette to the new leaves occurring during the chase period (T0 to T1)
was higher in asn1 mutant than in wild-type, as shown by the greater decrease of 15N% in rosette and the higher 15N% in new leaves of mutant. (C) Nitrogen
remobilization from the vegetative tissues to the seeds was monitored according to Diaz et al. (2008). 15N labelling was performed once at the vegetative stage. At
the end of the plant cycle, the dry weight of seeds and dry remains were recorded and used to calculate harvest index (HI, seed d. wt as a percentage of the
whole-plant d. wt). The amount of 15N remobilized from the rosette to the seeds was estimated through the calculation of 15N partitioning in seeds (P%15N;
15
N in seeds as a percentage of total 15N in the whole plant). Comparing P%15N/HI ratios facilitates comparison between plants with different HI. The
mutant (M) displayed significantly higher HI than wild-type (WT) and might be impaired for N-remobilization to the seeds as shown by its slightly lower
P%15N/HI ratio. Such preliminary finding needs confirmation using a second mutant allele.

studies of leaf senescence have shown that several aminotrans- nitrogen remobilization from old leaves to younger ones was
ferase and AS genes are also induced during senescence. In higher in the asn1 mutant than in the wild-type. Such a
sunflower, expression of two AS genes (HAS1 and HAS1.1) finding is in good agreement with the relationship between
detected only during leaf senescence, when asparagine severity of leaf-senescence and NRE at the vegetative stage
amounts increased, suggest a role of these enzymes in N remo- described by Diaz et al. (2008) and suggests that
bilization (Herrera-Rodriguez et al., 2006). N-remobilization from leaf to leaf does not depend only on
In Arabidopsis, among the three asparagine synthetase genes ASN1. By contrast, we found that 15N-remobilization from
ASN1, ASN2 and ASN3, only one is over-expressed during leaf rosette to seeds was slightly impaired in the asn1 mutant
senescence (Lam et al., 2003; Buchanan-Wollaston et al., (Fig. 4C). Although the partitioning of 15N in seeds (P%15N)
2005). A study of the asn1 mutant in our laboratory revealed did not differ between the asn1 mutant and the wild-type,
early senescing phenotypes (Fig. 4A). Although Lam et al. harvest index (HI; partitioning of dry matter in seeds) was sig-
(2003) reported that protein and amino acid concentrations nificantly higher for the mutant than wild-type. As a result, the
were increased in the seeds of ASN1 over-expressor lines, no ratio (P%15N/HI) appeared to be slightly lower in the mutant
significant difference between the wild-type and asn1 mutant than in the wild-type, indicating that ASN1 might have a role
could be found for seed protein concentration. The role of in harvest index and in N-remobilization from vegetative
ASN1 in nitrogen remobilization from leaf to leaf and from tissues to the seeds. This preliminary result shows that the
rosette to seeds was investigated using 15N tracing as described role of asparagine synthetase is certainly complex and further
by Diaz et al. (2008). Results presented in Fig. 4B show that investigations taking into account the contribution of the other
 Masclaux-Daubresse et al. — Nitrogen in agricultural plants 1149

asparagine synthetases, ASN2 and ASN3, are needed to under- and abiotic stresses through the induction of the GLN1,
stand the role of AS in nitrogen recycling and mobilization at GDH and ASN genes (Pérez-Garcia et al., 1998a, b; Olea
the whole-plant level. et al., 2004). Chaffei et al. (2004) showed that cadmium tox-
icity induced senescence-like symptoms and the expression of
the N remobilization enzymes GS1 and GDH in tomato leaves.
Phloem, a key factor for amino acid translocation from sources to
The induction of AS in roots might facilitate amino acid recy-
sinks
cling and storage of asparagine in this organ. The co-ordinated
Prior to phloem loading the central vacuole of mesophyll cells leaf N remobilization and root N storage is certainly essential
might be a site for transient storage of amino acids released from for plant recovery. Pageau et al. (2006) showed that GS1 and
protein degradation. In tobacco, the total amount of amino acids GDH genes in tobacco leaves responded to ethylene, jasmonic
exported from leaf blades increased five-fold during leaf ageing acid and several other plant defence elicitors as well as to virus
(Masclaux-Daubresse et al., 2006). Asparagine is the major and bacterial infections. N mobilization promoted by infection
translocated amino acid in pea. In cereals, tomato and tobacco, could be considered on the one hand as part of a
glutamine is the preferentially exported N-compound. In slash-and-burn strategy that should deprive the pathogen of
Arabidopsis, phloem sap mainly contains asparagine, glutamate nutrients by exporting nutrients away from the developing
and glutamine (J. Lothier, INRA, Versailles, France, unpubl. infection site, and on the other hand as a strategy to save nutri-
res.). During senescence, both asparagine and glutamine con- ents in healthy organs involved in recovery.
centrations increase in the phloem sap and both amino acids
are likely to play a key role in rendering nitrogen available for
R E G U L AT I ON OF N IT ROG E N U P TA KE ,
remobilization from the senescing leaf. The Arabidopsis
A S S IM I L AT I O N A N D R E M O B I L I Z AT I O N B Y
genome encodes 67 ( putative) amino acid transporters belong-
N I T R AT E AN D CAR B O N AVA I L A B I L I T I E S
ing to 11 gene families (reviewed by Rentsch et al., 2007).
The nature of the amino acid transporter involved in phloem N uptake by the roots and further N assimilation are integrated
loading during senescence is, however, poorly understood in the plant to match the nutrient demand of the whole organ-
(van der Graaff et al., 2006; for a review see ism. External stimuli or stresses as well as nutritional status of
Masclaux-Daubresse et al., 2008). the plant modulate the expression and/or the activity of trans-
port systems and enzymes by various regulatory mechanisms.
The first mechanism operates at the transcriptional level and
N-storage and remobilization are essential for plant survival
includes the induction by the substrates and the repression
N-storage and remobilization potential are important for exerted by endogenous N assimilates. This results in an upre-
both annual and perennial plants. For annual plants, as men- gulation when N is low and a downregulation when N is high.
tioned above, nitrogen remobilization is important for seed Accordingly, several NRT2 and AMT1 transporters as well as
production and seed nitrogen content. Nitrogen content in Nia and Nii genes were found to be transcriptionally repressed
the seeds further determines germination efficiency and survi- by N metabolites such as amino acids like glutamine (Tsay
val of young seedlings. et al., 2007; Meyer and Stitt, 2001). On the other hand, in
Nitrogen remobilization is also important for perennial plant response to N deprivation, expression of many ammonium
survival. Trees, which grow in low nitrogen environments and high-affinity nitrate transporters is induced or repressed
most of the time, have two phases of nitrogen remobilization. (reviewed by Tsay et al., 2007). In response to N deprivation,
Nitrogen is remobilized from the senescing leaves in autumn expression of GLN and GDH genes is also up- or down-
to be stored in trunks during winter. N is remobilized a regulated. In tobacco, it was shown that ammonium and
second time from trunks to developing organs in spring amino acids regulate GLN and GDH transcript levels
before root N uptake becomes the main process to meet tree (Masclaux-Daubresse et al., 2005). Glutamate feeding over
N needs. As trees age, the internal cycling of N becomes 5 h increased GLN1 mRNA while both glutamate and
more and more important in the whole-tree N-budget. Both proline feeding decreased GLN2 mRNA. Ammonium,
nitrogen withdrawal from senescing leaves and root N uptake proline, glutamine and glutamate increased GDH transcripts.
contribute to the build-up of N storage pools and to the effi- Effects of N-metabolites on GLN and GDH transcript levels
cient nitrogen management that are essential for plant survival proved to be sensitive to calcium blockers and the K252a
over years (Millard et al., 2006, 2007). protein-kinase inhibitor. Evidence showed that ammonium
Forage grasses are subject to frequent defoliation by herbi- itself regulates GLN genes at the transcriptional level. In
vores or mechanical harvesting. Recovery of grasses after soybean, co-operation between three distinct promoter
defoliation is related to the availability of carbon and nitrogen regions is necessary for ammonium-stimulated expression of
reserves in the remaining tissues (Volenec et al., 1996). the GS15 gene. The interaction among these regions may be
Decreasing mineral N supply before defoliation was shown facilitated by an HMG A (high-mobility group A)-like
to decrease the availability of N reserves in leaves and as a protein that binds to the proximal and distal promoter
result the absolute amount of N subsequently remobilized to regions of the soybean GS15 gene (Reisdorf-Cren et al.,
roots. 2002, and references therein). Global transcriptome studies
Interestingly, it was shown that N remobilization and senes- after nitrate induction (Scheible et al., 2004) confirmed regu-
cence can be induced prematurely by environmental factors, lation of N uptake and assimilation by nitrate at the expression
such as pathogen attack or heavy metals. Evidence corrobo- level and showed a large action spectrum of nitrate as a regu-
rates that the N remobilization process is enhanced by biotic lator of gene expression, co-ordinating for example C and N
1150 Masclaux-Daubresse et al. — Nitrogen in agricultural plants

metabolism. Using NR mutants, it was shown that much of this AtNRT1.1 functions as a high-affinity transporter whereas it
regulation is exerted by nitrate itself (Wang et al., 2004). is active in the low affinity range when dephosphorylated
The stimulation of N uptake and N assimilation by photo- (for a review see Tsay et al., 2007). Tsay and co-workers
synthesis (for a review see Lillo, 2008) ensures that N obtained significant insight into the role of the CIPK23
uptake is correlated with C status. For example, nitrate kinase in the specific phosphorylation of the AtNRT1.1
uptake and reduction are co-ordinately regulated by a circadian protein in response to nitrate levels, demonstrating
control. This control has often been attributed to the regulatory AtNRT1.1-mediated sensing in the primary nitrate response
action on gene expression of sugars produced by photosyn- (Ho et al., 2009).
thesis and transported downward to the roots. This has been The best studied post-translational regulation in N metab-
shown for the ammonium and nitrate transporters, NR and olism is the regulation of NR in higher plants. NR is inacti-
NiR. The regulation of nitrate uptake and transporters seems vated by a two-step process that involves the phosphorylation
to be independent of the known sugar regulation pathways, of ser543, as shown in spinach, followed by the binding of an
such as hexokinase signalling (Lillo, 2008). Wirth et al. inhibitory 14-3-3 protein kinase. In addition, both CDPK
(2007) showed that upregulation of nitrate transporters (calcium-dependent protein kinases) and SnRK1 protein
(AtNRT2.1 and AtNRT1.1) was related to the concentration kinases are able to phosphorylate NR at least in vitro (reviewed
of glucose 6-phosphate. In contrast, the diurnal regulation of by Lillo, 2008). When a modified form of NR, no longer sus-
Nia transcripts is governed not only by sugars but also by ceptible to post-translational dark inactivation, was over-
light regulation via phytochrome (Lillo, 2008). In addition, it expressed, the resulting protein did not decline in the second
was observed that Nia expression is controlled by signals part of the photoperiod. The inactive phosphorylated form is
from photosynthetic electron flow, which adds a new facet to re-activated by dephosphorylation, probably by PP2A.
the intracellular cross-talk between chloroplasts and the Moreover, evidence showed that there is a correlation
nucleus (Lillo, 2008). between the phosphorylation state or the activation state of
HY5 and its homologue HYH, two transcription factors NR and the rate at which NR protein decreases.
from the bZIP family, are essential for phytochrome- Plastidic glutamine synthetase from Medicago truncatula is
dependent light-activated expression of NR (Lillo, 2008). also regulated through phosphorylation and 14-3-3 inter-
ChIPchip analyses showed a binding site for HY5 in the actions. The GS2 phosphorylation site ser97, critical for the
Nia2 promoter (Lillo, 2008). The NRT1.1 promoter also has interaction with 14-3-3 and subsequent proteolysis, was ident-
three binding sites for HY5, although HY5 seems to have a ified by directed mutagenesis. Cytosolic glutamine synthetases
negative effect on transcription in this case (Lillo, 2008). from M. truncatula are also regulated by phosphorylation but
Castaings et al. (2009) identified NLP7 as a regulator of Nia by calcium-independent kinases (Lima et al., 2006, and refer-
expression in Arabidopsis. Arabidopsis nlp7 mutants are ences therein). Phosphorylation occurs at more than one
defective in the nitrate induction of Nia genes and NRT2.1 residue and increases affinity for the substrate glutamate.
and NRT2.2. Interestingly, mutants in the CIPK8 gene, In addition to phosphorylation, several chloroplastic
which encode a protein kinase (Hu et al., 2009), are also enzymes of nitrogen assimilation such as NIR, GS2 and
unable to fully induce expression of several genes by nitrate, Fd-GOGAT are also redox-regulated through the thioredoxin
such as the Nia genes, NRT2.1, NRT1.1 and several others. It system (for reviews see Lemaire et al., 2007; Lillo, 2008).
is tempting to speculate that CIPK8 might be involved in the
same regulation pathway as NLP7. NLP7 belongs to a gene
T R A NS GE N IC E F FO R T S TO M AN IP U L AT E NU E
family with nine different members, but the functions of the
other NLP proteins are still unknown. With the aim of improving NUE, many critical candidate
In Arabidopsis, compelling evidence shows that SnRK1s genes have been manipulated, over-expressing them or using
(Snf1-related protein kinases) are central integrators of tran- knockout mutations, in order to test their effects on biomass
scription networks in plant stress and energy signalling that and plant nitrogen status. Several good reviews have been
are inactivated by sugars (Baena-Gonzalez et al., 2007). written on this subject that provide more detail than mentioned
SnRK1 proteins were shown to specifically regulate the in this section (Andrews et al., 2004; Good et al., 2004; Pathak
ASN1 gene, encoding the dark-induced asparagine synthetase et al., 2009).
also known as DIN6. ASN1/DIN6 is induced by darkness, Until now, probably because of strong post-transcriptional
hypoxia, DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] controls (see above), manipulating nitrate uptake through the
and other stress within 3 – 6 h. The protein kinase inhibitor over-expression of HATS-like NRT2.1 led to increased
K252a abolishes such induction and the two ubiquitously nitrate influx under some conditions but did not change the
expressed members of the SnRK1 group, Kin10 and Kin11, phenotypic NUE or nitrate utilization (Fraisier et al., 2000).
specifically activate a DIN6promoter::LUC fusion. Mutation NR has long been considered to be the rate-limiting step in
of the G-box (CACGTG, G1) proximal to the TATA box abol- nitrate assimilation. The utility of transgenic over-expression
ished most of the activation of DIN6promoter::LUC by of NR/NiR for major improvements of NUE, however,
KIN10, hypoxia, darkness and DCMU. remains uncertain. Nicotiana tabaccum plants constitutively
Rapid post-translational regulation such as protein modifi- expressing NR from N. plumbaginifolia showed delay in
cation is the second mechanism that controls nitrogen uptake NR-activity loss under drought, which allowed them to
and assimilation. Post-transcriptional regulation of nitrate present a more rapid recovery after short-term water deficit
transporters by phosphorylation has recently been described (Ferrario-Mery et al., 1998). Then, under field conditions of
for the nitrate transporter NRT1.1. When phosphorylated, fluctuating water availability, constitutive NR expression may
 Masclaux-Daubresse et al. — Nitrogen in agricultural plants 1151

confer some physiological advantage. Over-expressing NR or In addition to manipulating enzymes involved in nitrogen
NiR in Arabidopsis, potato or tobacco reduced nitrate levels assimilation of amino acid metabolism, the generation of
in plant tissues but did not increase biomass yield, tuber plants modified for the expression of transcription factors
numbers or seed yields. Over-expression of Nia or Nii genes has also been attempted. For example, ectopic expression of
in plants increased mRNA levels and often affected N the maize Dof1 transcription factor, which regulates the
uptake without modifying yield or plant growth regardless of expression of genes involved in organic acid metabolism, led
the nitrogen source available. This is believed to be due in in Arabidopsis to the accumulation of amino acids and to an
part to the complex post-transcriptional regulation of NR increase of growth under N-limiting conditions. These
(reviewed by Pathak et al., 2009). effects suggest that NUE could also be improved by manipu-
Over-expression of cytosolic glutamine synthetase GS2 lating carbon metabolism pathways. PII-like, NLP7 and TOR
genes was performed in N. tabaccum and Oryza sativa using (target of rapamycin) proteins, which are potentially linked
the Rubisco small subunit promoter and the CaMV 35S pro- to C and N sensing in plants, are other candidates for further
moter, respectively (Hoshida et al., 2000; Migge et al., engineering as shown by the increased plant growth, yield
2000). In N. tabaccum, over-expression enhanced growth rate and stress resistance acquired by TOR-overexpressing plants
and in O. sativa it increased photorespiration and drought tol- (Ferrario-Mery et al., 2006; Deprost et al., 2007; Castaings
erance. Attempts to over-express GS1 genes are more numer- et al., 2009).
ous and have used different promoter combinations, Hibberd et al. (2008) suggested that engineering C4 rice or
including CaMV 35S, RolD and small Rubisco subunit C4 wheat would be a good way to improve NUE. The Rubisco
(rbcS). Effects on plant biomass and grain yield were also protein is known to be used as a storage protein in C3 herbac-
more successful. For example, over-expression of the eous plants and trees (Millard et al., 2006). In elevated atmos-
Phaseolus vulgaris GS1 gene under the control of the rbcS pheric CO2, Rubisco carboxylase activity is increased and
promoter in wheat resulted in significantly higher root and Rubisco protein content is decreased. The selective loss of
grain yield with higher N content in grain in some lines Rubisco enzyme under elevated CO2 thus benefits NUE
(Habash et al., 2001). Over-expression of the Pisum sativum without necessarily significantly changing the leaf C assimila-
GS1 gene under the control of the CaMV 35S promoter in tion rate. The lower Rubisco levels in C4 plants explains why
N. tabaccum resulted in biomass and leaf protein increases NUE is higher in C4 crops than in C3.
(Oliveira et al., 2002). More recently, over-expressing a GS1
isoenzyme of maize in maize under the control of the
NATUR AL VA RI ATI ON OF N ITROG EN U PTA KE
CsVMV promoter increased kernel number and kernel size,
A ND NI T ROG E N RE M OBI L I Z AT I ON IN
thus increasing yield by 30 % [ patent AU2007306040 (A1);
A R A B I DO P S IS
http://v3.espacenet.com/publicationDetails/biblio?CC=AU&
NR=2007306040A1&KC=A1&FT=D&date=20080417&DB= More than 300 accessions of Arabidopsis, originating from
EPODOC&locale=en_EP]. In summary, several studies have various locations worldwide, are available in stock centres.
demonstrated a direct correlation between an enhanced GS Probably due to a selective adaptation to original edaphic
activity in transgenic plants and biomass or yield (Good and climatic environments, they show natural variation of
et al., 2004, and references therein). Although over-expression their development and they constitute large genetic and pheno-
of GOGAT genes has been rare, Yamaya et al. (2002) reported typic resources (McKhann et al., 2004). Several recent papers
a spectacular effect of the over-expression of NADH-GOGAT have presented the first evidence that natural variation exists
under the control of its own promoter in rice. Transgenic plants for nitrogen metabolism, including nitrogen uptake and nitro-
showed an increase (up to 80 %) in grain weight. In con- gen remobilization. The first clue was provided by the analysis
clusion, studies show that over-expression of GS or GOGAT of root plasticity. Studies in Arabidopsis have shown the stimu-
genes can improve biomass and grain yields depending on lation of root growth by a localized source of nitrate
which gene allele and which promoters are used. This indi- (Robinson, 1994; Forde and Lorenzo, 2001). Walch-Liu and
cates that further characterization is required to demonstrate Forde (2008) assayed the extent of root stimulation using a
the beneficial effects of such strategies for crops and in field small collection of six accessions. A large range of responses
conditions. was obtained from one extreme, which was not significantly
Attempts to over-express AS were carried out in tobacco and affected, to the other extreme that showed a 90 % increase of
Arabidopsis (for a review see Good et al., 2004). Interestingly, primary root growth. This observed variation of root adap-
over-expression of ASN1 in Arabidopsis enhanced soluble seed tation to nitrogen availability should have consequences for
protein content and total protein and increased fitness of plants nitrogen uptake in plants.
grown under nitrogen-limiting conditions (Lam et al., 2003). Our recent investigation of natural variation in nitrate uptake
Alanine is a major amino acid for nitrogen storage under and nitrogen remobilization in Arabidopsis gives the second
anaerobic stress such as flooding. Over-expression of barley clue (Fig. 5; C. Masclaux-Daubresse and F. Chardon,
alanine amino transferase under the control of root promoters unpubl. res.). A core-collection of 18 accessions of
in canola and rice had interesting effects, considerably increas- Arabidopsis was grown under limiting and ample nitrogen
ing plant biomass, seed yield, NUE and shoot nitrogen concen- nutritive conditions. The aim of this study was to collect
tration when plants were grown at low nitrate supply (Good data allowing us to monitor the natural variation of N uptake
et al., 2007; Shrawat et al., 2008). These results are of particu- at the vegetative stage and the N-remobilization to the seeds
lar interest, showing that it is possible to improve NUE by at the reproductive stage depending of nitrogen availability,
manipulating downstream steps in N-remobilization. and also to measure traits related to NUE such as biomass of
1152 Masclaux-Daubresse et al. — Nitrogen in agricultural plants

Col-0 Sha Stw-0 Bur-0 Tsu-0

DW DW DW DW DW
4 4 4 4 4
15 2 15 2 15 2 15 2 15 2
NHI N% NHI N% NHI N% NHI N% NHI N%
0 0 0 0 0
–2 -
–2 –2 –2 –2
–4 –4 –4 –4 –4
HI NupE HI NupE HI NupE HI NupE HI NupE

%NDR %NSEED %NDR %NSEED %NDR %NSEED %NDR %NSEED %NDR %NSEED

F I G . 5. Nitrogen absorption and nitrogen remobilization profiling of five Arabidopsis accessions. A core collection of 18 accessions of Arabidopsis grown with
10 mM nitrate was used to measure traits related to biomass, N uptake, N remobilization and NUE (C. Masclaux-Daubresse and F. Chardon, unpubl. res.). Five
accessions representative of the main classes found are presented. Traits measured at the vegetative stage (40 d after sowing) are rosette dry weight (DW), nitro-
gen concentration in the rosette (N%) and N-uptake efficiency (NupE) as the quantity of 15N absorbed at 40 d after sowing divided by plant biomass. Traits
measured at the end of the plant cycle are N remobilization efficiency (15NHI) expressed as the partitioning of 15N in seeds compared with whole plant
(seeds þ dry remains; see Diaz et al., 2008), harvest index (HI) expressed as the partitioning of biomass (dry matter) in seeds (DM of seeds/DM of the
whole plant), N concentration in dry remains (%NDR) and N concentration in the seeds (%NSEED). Individual values ranged between [24; þ 4] and centred
around the mean value of the core collection (value 0). The small sample of accessions highlights the variation of performances. Plants, such as Col0 or
Sha, have a relatively good N uptake. The highest N remobilization score was found in Stw-0 while plants with high N percentage and high biomass were
Bur-0 and Tsu-0.

rosettes at the vegetative stage, seed yield, harvest index and characterization of edaphic environments and the metabolism
nitrogen concentrations in the different plant material collected of different Arabidopsis accessions would consequently
(at vegetative and reproductive stages). Using all the data col- allow a better understanding of how these modules are regu-
lected at low and high nitrogen supply, groups of accessions lated according to the nitrogen availability in soil.
can be clustered, assembling plants that have similar responses Natural variation also exists in crops. Approaches currently
depending on nitrate availability. Surprisingly, for most of the performed by breeders to improve varieties for many agrono-
traits measured or computed the variation was higher at high mical traits include QTL mapping and marker-assisted selec-
nitrate supply when N uptake and N remobilization are not tion. However, it is worth noting that the experiments
forced by nitrate limitation. Figure 5 shows schematically performed on Arabidopsis presented above have been carried
the large variation observed between the classes of accessions. out on wild plants, meaning that the plants studied have not
The differences between the lowest and highest performing been modified for agronomic criteria and that no adaptive
accessions were three-fold for N uptake and six-fold for N selection has decreased differences between them. Using
remobilization. We also noted interesting correlations Arabidopsis rather than highly selected plants such as crops
between how plants manage N uptake and N metabolism for such approaches should then be more informative.
and their biomass. Considering the relative NUE at the vegeta- Natural variation of N uptake and N remobilization identified
tive stage as the ratio DW/N% (optimal NUE: plants with large in the model plant Arabidopsis is a source of knowledge that
biomass and low nitrogen concentration), we found that the can be useful to transfer to crops.
estimated NUE in the core-collection was not different at lim-
iting and ample nitrogen supply, suggesting that NUE at the
QU AN TITATIV E GEN ETI C A ND GEN ETI C
vegetative stage was determined solely genetically (C.
B A S E S O F NI T ROG E N U P TA K E , A S S I M I L AT I O N
Masclaux-Daubresse and F. Chardon, unpubl. res.).
A ND RE M O B I L IZ AT IO N
The last clue is provided by the Arabidopsis eFP-Browser
database, which combines microarray analyses (Winter et al., QTLs for NUE and other agronomic traits have been mapped
2007). Some experiments included in this database used in numerous plant species, including maize, rice and
several accessions (Lempe et al., 2005). It is possible to Arabidopsis (Hirel et al., 2001; Obara et al., 2001; Loudet
select specifically the pattern of genes involved in primary N et al., 2003). QTL mapping for nitrogen-related enzyme activi-
metabolism. Although plants were cultivated in the same con- ties such as nitrate reductase or glutamine synthetase are rarer.
ditions (4-day-old seedlings grown in soil in the glasshouse), Even more rare is the mapping of N-remobilization or N-influx
the signal intensities of some N genes indeed varied among QTLs because of the difficulty in performing 15N tracing on
these accessions (Fig. 6), revealing the existence of genetic large populations.
variation for the transcription level of N genes. Whether The first report of mapping QTL for N remobilization was in
such variation is correlated with N-dependence and NUE barley using the N-balance method, which requires monitoring
remains to be determined. the difference in flag leaf N-content between anthesis and
Experiments presented above provide ideas about the maturity [DN (mg) per leaf ] (Mickelson et al., 2003). QTLs
various traits that can be measured to explore NUE (N-gene explaining the variation of this trait were mapped on chromo-
transcription levels, N uptake efficiency, NRE, nitrogen somes 6 and 7. Mickelson et al. (2003) also mapped two QTLs
content, enzyme activities, biomass) and encourage computing for grain protein concentration on chromosomes 2 and 6. There
data according to a systems biology approach, in order to was no co-localization between the QTLs for DN per leaf and
reveal the functioning of the different modules that constitute grain protein content. However, the most prominent QTL for
nitrogen metabolism adaptation in plants. A better grain protein content on barley chromosome 6 appeared to
 Masclaux-Daubresse et al. — Nitrogen in agricultural plants 1153

Gene Bay-0 C24 Col-0 Cvi-1 Est Kin-0 Ler-2 Nd-1 Sha Van-0
NRT2.1
NIA1
NIA2
NIR
ASN1
GDH1
GDH2
GLU2
GS1.1

F I G . 6. Heat map illustrating the natural variation in expression of genes involved in nitrogen metabolism. Signal levels of N genes in ten accessions were
obtained from the Arabidopsis eFP-Browser. For each gene, high expression is depicted as dark shading, and low expression is depicted as light shading.

be a potential homologue of the grain protein QTL from durum were also found in eight clusters, while N-remobilization
wheat mapped by Joppa et al. (1997). Recently, a wheat QTL QTLs mainly coincided with leaf senescence QTLs.
was cloned through positional cloning and fine mapping (Uauy Co-localization with N-related genes showed that the two
et al., 2006). The locus encodes an NAC transcription factor, NR loci (chromosomes 1 and 4, see Hirel et al., 2001)
NAM-B1, which accelerates leaf senescence and increases coincided with QTLs affecting kernel number at high N
nutrient filling in developing grains. The ancestral wild input and kernel weight at both low and high N. Grain
wheat allele is functional whereas modern wheat varieties yield-related traits coincided with the three GS1 loci corre-
carry a non-functional NAM-B1 allele. The role of NAM-B1 sponding to Gln1-2, Gln1-3 and Gln1-4. The GS2 locus
was confirmed by reducing the RNA levels of multiple (chromosome 10) coincided with a leaf senescence QTL
NAM homologues by RNA interference, resulting in delayed (Coque et al., 2008).
senescence (by more than 3 weeks) and reduced wheat grain QTLs for NUE and N-enzyme activities were also recently
protein, zinc and iron contents (by more than 30 %). The explored in wheat (Habash et al., 2007; Laperche et al., 2007;
effect of the chromosome Gpc-B1 region including the Fontaine et al., 2009). Interestingly, Habash et al. (2007)
NAM-B1 gene was studied further by introgressing the showed that QTLs for GS activity were invariably co-localized
Gcp-B1 locus in hexaploid near-isogenic lines. As a result of with those for grain N, and that increased activity was associ-
Gcp-B1 introgression, significantly lowered straw N concen- ated with higher grain N. This finding was confirmed by
tration at maturity and higher nitrogen harvest index (NHI) Fontaine et al. (2009) on another population. Unlike in
were measured, suggesting that the functional Gcp-B1 allele maize, there was no correlation between GS activity and
improves N remobilization and diminishes the amount of nitro- yield components in wheat.
gen lost in residual dry remains (Brevis and Dubcovsky,
University of California, Davis, CA, unpubl. res.).
Comparing NAM-B1 knockdown RNAi and control lines
CO NC L US IO NS
showed similar results (Waters et al., 2009).
In the same barley population used by Mickelson et al. Improving global plant productivity and product quality
(2003) to determine grain protein and leaf N storage and remo- together with taking care of environmental quality and
bilization, QTLs for leaf amino-, carboxy- and endo-peptidase human wellbeing are the main challenges for the immediate
activities were determined (Yang et al., 2004). QTL future (Vitousek et al., 2009). Such a goal depends on agricul-
co-localization strongly suggested that the major endo- or tural development and policy and can be achieved by provid-
amino-peptidases were not involved in leaf N remobilization ing the right nutrient source at the right rate, the right time
or in the control of grain protein content. By contrast, QTL and the right place. To improve sustainable agricultural pro-
co-localization suggested that vacuolar carboxy-peptidase iso- duction, it is also necessary to grow crops that can remove
enzymes are involved in leaf N remobilization. the nutrient applied to soil efficiently, and therefore require
More recently, N-uptake and N-remobilization QTLs have less fertilizer. Such global ‘resource use efficiency’ necessi-
been mapped in maize (Coque and Gallais, 2007; Coque tates having a global view of plant physiology, plant uptake
et al., 2008). The authors monitored an impressive number capacity, plant metabolism and plant response to restrictions,
of traits for NUE, leaf senescence, enzyme activities, yield, as well as a view of soil physical and chemical properties.
biomass, N uptake and N remobilization, which, together This review gives an overview of the different metabolic and
with the former data from Hirel et al. (2001) and additional physiological clues that agronomical research has provided.
gene positional cloning, provide a huge amount of information The enzymes and regulatory processes that can be manipulated
about the coincidence of agronomical, physiological, bio- to control NUE are presented. The last results obtained from
chemical and genomic traits. The study by Coque et al. natural variation and QTL studies show the complexity of
(2008) is also the first and to date the only study to have NUE and open new perspectives. With regard to the complex-
used the 15N tracing method to map N-remobilization QTLs. ity of the challenge we have to face and with regard to the
QTL clustering showed an antagonism between N remobiliza- numerous approaches available, the integration of data
tion and N uptake at several loci. Positive coincidences coming from transcriptomic studies, functional genomics,
between N uptake, root system architecture and leaf greenness quantitative genetics, ecophysiology and soil science into
1154 Masclaux-Daubresse et al. — Nitrogen in agricultural plants

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