Field Crops Research 123 (2011) 139–152

Contents lists available at ScienceDirect

Field Crops Research

journal homepage: www.elsevier.com/locate/fcr

Identification of traits to improve the nitrogen-use efficiency of wheat genotypes

O. Gaju a , V. Allard b,c , P. Martre b,c , J.W. Snape d , E. Heumez e , J. LeGouis b,c , D. Moreau b,c,1 , M. Bogard b,c ,
S. Griffiths d , S. Orford d , S. Hubbart a , M.J. Foulkes a,∗
a
Division of Plant and Crop Sciences, School of Biosciences, University of Nottingham, Leicestershire, LE12 5RD, UK
b
INRA, UMR 1095 Genetics, Diversity and Ecophysiology of Cereals, Clermont-Ferrand, F-63 100, France
c
Université Blaise Pascal, UMR 1095 Genetics, Diversity and Ecophysiology of Cereals, F-63 177 Aubière, France
d
John Innes Centre, Norwich Research Park, Colney Norwich NR4 7UH, UK
e
INRA/USTL, UMR 1091 Abiotic Stress and Plant Development, Mons-Péronne, F-80 200, France

a r t i c l e i n f o a b s t r a c t

Article history: Nitrogen (N) fertilizer represents a significant cost for the grower and may also have environmental
Received 2 November 2010 impacts through nitrate leaching and N2 O (a greenhouse gas) emissions associated with denitrification.
Received in revised form 8 May 2011 The objectives of this study were to analyze the genetic variability in N-use efficiency (grain dry matter
Accepted 9 May 2011
(DM) yield per unit N available from soil and fertilizer; NUE) in winter wheat and identify traits for
improved NUE for application in breeding. Fourteen UK and French cultivars and two French advanced
Keywords:
breeding lines were tested in a 2 year/four site network comprising different locations in France and in
Nitrogen-use efficiency
the UK. Detailed growth analysis was conducted at anthesis and harvest in experiments including DM
Nitrogen-utilization efficiency
Canopy senescence
and N partitioning. Senescence of either the flag leaf or the whole leaf canopy was assessed from a visual
Wheat score every 3–4 days from anthesis to complete canopy senescence. The senescence score was fitted
Breeding against thermal time using a five parameters monomolecular-logistic equation allowing the estimation
of the timing of the onset and the rate of post-anthesis senescence. In each experiment, grain yield was
reduced under low N (LN), with an average reduction of 2.2 t ha−1 (29%). Significant N × genotype level
interaction was observed for NUE. Crop N uptake at harvest on average was reduced from 227 kg N ha−1
under high N (HN) to 109 kg N ha−1 under LN conditions while N-utilization efficiency (grain DM yield per
unit crop N uptake at harvest; NUtE) increased from 34.0 to 52.1 kg DM kg−1 N. Overall genetic variability
in NUE under LN related mainly to differences in NUtE rather than N-uptake efficiency (crop N uptake
at harvest per unit N available from soil and fertilizer; NUpE). However, at one site there was also a
positive correlation between NUpE and NUE at LN in both years. Moreover, across the 2 year/four site
network, the N × genotype effect for NUpE partly explained the N × genotype effect for grain yield and
NUE. Averaging across the 16 genotypes, the timing of onset of senescence explained 86% of the variation
in NUtE amongst site-season-N treatment combinations. The linear regression of onset of senescence
on NutE amongst genoytpes was not significant under HN, but at three of the four sites was significant
under LN explaining 32–70% of the phenotypic variation amongst genotypes in NutE. Onset of senescence
amongst genotypes was negatively correlated with the efficiency with which above-ground N at anthesis
was remobilized to the grain under LN. It is concluded that delaying the onset of post-anthesis senescence
may be an important trait for increasing grain yield of wheat grown under low N supply.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Nitrogen-use efficiency (NUE) can be defined as the grain dry

Abbreviations: AD, anthesis date; GPC, grain protein concentration; GY, grain
yield; AGNA , above-ground N at anthesis; AGNH , above ground N at harvest;
NUE, nitrogen-use efficiency; NUpE, nitrogen-uptake efficiency; NUtE, nitrogen-
utilization efficiency; NHI, nitrogen harvest index; NRE, crop N remobilization
efficiency.
∗ Corresponding author. Tel.: +44 0 1159 516024; fax: +44 0 1159 516060.
E-mail address: John.Foulkes@nottingham.ac.uk (M.J. Foulkes).
1
Present address: INRA, UMR1210 Biologie et Gestion des Adventices, F-21 065
Dijon, France.
matter (DM) yield (kg DM ha−1 ) divided by the supply of avail-
able nitrogen (N) from the soil and fertilizer (kg N ha−1 ; Moll et al.,
1982). Improving NUE is an important target because it increases
profitability, either through greater yields or reduced fertilizer
costs. Improving NUE will also reduce the greenhouse gas (GHG)
emissions associated with the production of each kg of yield. For
example, it has been estimated that N fertilizer accounts for more
than 70% of the GHGs associated with the production of wheat
(Mortimer et al., 2004). It is also likely that improving NUE will

0378-4290/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.fcr.2011.05.010
140 O. Gaju et al. / Field Crops Research 123 (2011) 139–152
reduce the risk of nitrate leaching by reducing the amount of fer-
tilizer that is not used by the crop. Winter wheat grown in the
UK has been estimated to have an average N recovery of 65%
and a NUE of 25 kg DM kg−1 N available (Sylvester-Bradley and
Kindred, 2009) and based on the same assumptions for the grain
N–total N ratio and available soil N, very similar results are obtained
for France (LeGouis, per. comm.). Therefore, there is increasing
emphasis worldwide in breeding wheat cultivars with potential
for improving NUE and for reducing excessive input of fertilizers,
while maintaining an acceptable yield (Hirel et al., 2007; Foulkes
et al., 2009).
Nitrogen-use efficiency can be further divided into two compo-
nents: (i) N-uptake efficiency (crop N uptake per unit N available
from the soil and fertilizer; NUpE) and (ii) N-utilization efficiency
(grain DM yield per unit crop N uptake at harvest; NUtE). Genetic
gains in NUE with breeding estimated in experiments examin-
ing historic sets of cultivars under low N supply were related
mainly to improvements in NUpE in spring wheat in Mexico (Ortiz-
Monasterio et al., 1997) and Finland (Muurinen et al., 2006) and to
NUtE in winter wheat in France (Brancourt-Hulmel et al., 2003)
and the UK (Foulkes et al., 1998). Under high N supply, studies
in the UK (Foulkes et al., 1998), Mexico (Ortiz-Monasterio et al.,
1997) and Finland (Muurinen et al., 2006) found that genetic gains
in NUE were explained approximately equally by NUpE and NUtE.
In an investigation of 20 winter wheat cultivars in France, NUpE
accounted for more of the genetic variation in NUE at low N than
at high N (LeGouis et al., 2000). In contrast, for 12 spring wheats
in California, NUpE was the most important component of NUE
at both low and high N supply (Dhugga and Waines, 1989). In an
examination of 39 cultivars at 5 N rates in the UK, cultivar differ-
ences in N uptake were only found at the highest 3 N rates, and
NUtE explained more of the variation in grain yield than NUpE at
all 5 N rates (Barraclough et al., 2010). Overall these investigations
tended to show that effects of N supply and genotype on yield
often interact. Furthermore, at low N supply the yield could be
more closely correlated with either NUpE or NUtE depending on
location, whereas at high N supply yield was usually more closely
correlated with NUtE than NUpE. Similar observations to those of
Ortiz-Monasterio et al. (1997) and LeGouis et al. (2000) for NUpE
to be more closely correlated to NUE at low N than at high N have
been made in other species including barley (Anbessa et al., 2009)
and oilseed rape (Horst et al., 2003), although in maize NUpE was
more closely correlated to NUE at high N and NUtE at low N (Bertin
and Gallais, 2000). Most cultivar testing systems worldwide use a
relatively high N supply, which means that plant breeders select
cultivars that perform well under these conditions. If genotype and
N supply do interact as indicated by the above-mentioned investi-
gations then this means that cultivars selected for high yield under
high N conditions may not be the highest yielding under lower N
conditions. In order to reliably identify which cultivars will yield
well at lower N supplies will require both breeding and testing at
low levels of N input (Brancourt-Hulmel et al., 2005) or an under-
standing of which plant traits are important for yield under these
conditions.
One of the key traits that could explain the genetic variability
associated with yield variation under low N is leaf senescence kinet-
ics. The capacity of a genotype to retain green leaf area for longer
than a standard genotype during grain-filling has been referred to
as the ‘stay-green’ phenotype (Thomas and Smart, 1993). Under
optimal conditions, wheat crops are in general little limited by the
assimilate supply during grain filling (Dreccer et al., 1997; Borrás
et al., 2004; Calderini et al., 2006), but under low to moderate N fer-
tilizer levels yields may be more limited by post-anthesis assimilate
supply. Delaying senescence should provide more carbohydrate
for above-ground growth by prolonging photosynthesis, but also
should provide more carbohydrate to roots for prolonged N uptake
(Dreccer, 2005). So stay green genotypes may have an increased
source:sink ratio during grain filling with an increased fraction of
N derived from the soil N. Using stay green genotypes under low
N was associated with improved performance in both sorghum
(Borrell and Hammer, 2000) and maize (Mi et al., 2003). Genetic
variation in functional stay-green (delayed senescence associated
with extended photosynthesis) lines has been reported in wheat
(Silva et al., 2000; Verma et al., 2004; Foulkes et al., 2007), although
the underlying physiological mechanisms have not been studied
extensively. Christopher et al. (2008) found that the stay-green phe-
notype in spring wheat SeriM82 was associated with extraction of
deep soil water in Australia. In sorghum hybrids, stay-green has
been linked to changes in the balance between N demand and sup-
ply during grain filling resulting in a slower rate of N translocation
from the leaves to the grain compared with senescent genotypes
(Borrell and Hammer, 2000; Van Oosterom et al., 2010b). In sum-
mary, the maintenance of a photosynthetically active canopy in the
post-anthesis period under low to moderate N supply should opti-
mize the efficiency of N input use for grain DM production, although
in wheat cultivars used for bread-making any trade-off with grain
protein concentration (GPC) with regard to the ca. 13% threshold
for bread-making must also be considered.
Increased economic cost and environmental concerns have
increased the need to manage fertilizer use more judiciously. These
pressures will be exacerbated by increasing demand for food and
fuel as the human population increases. It is clear that understand-
ing is required about which traits determine yield under low N
conditions in order to increase NUE and reduce the use of N fer-
tilizer, while maintaining or increasing yield, yet relatively few
studies have investigated this. This study uses a set of field experi-
ments conducted in contrasting pedo-climatic conditions in France
and the UK to understand the physiological mechanisms and iden-
tify the traits which determine differences between cultivars in
the ability to maintain yield when grown under low N supply.
The study tests the hypotheses that: (i) crop NUtE is an impor-
tant determinant of yield differences between cultivars grown at
low to moderate N levels and (ii) the physiological basis of genetic
variation in NUtE is partially explained by senescence kinetics in
the post-anthesis period. It was hoped that information on traits
would provide selection criteria for breeding and cultivar choice in
low N environments in northern Europe and worldwide in future
years.
2. Materials and methods
2.1. Sites and experimental treatments
Field experiments were carried out at two sites in the UK
and two sites in France in 2006–7 and 2007–8; University of
Nottingham, Sutton Bonington, UK (52◦ 50 N, 1◦ 14 W), John Innes
Centre, Norwich, UK (52◦ 38 N, 1◦ 18 E), INRA Estrées-Mons, France
(49◦ 08 N, 03◦ 00 E) and INRA Clermont-Ferrand, France (45◦ 46 N,
03◦ 09 E). Information on soil types is given in Table 1. The same
experimental treatments were tested in the eight site-seasons in
which 14 cultivars and two advanced breeding lines were grown at
two rates of N fertilizer (Table 1). Each experiment at Sutton Bon-
ington (SB) and Clermont-Ferrand (CF) used a split-plot design in
which N treatment was randomized on main plots, genotypes were
randomised on the sub-plots and each treatment was replicated
three times. Sub-plot size was 24 × 1.65 m at SB and 24 × 1.5 m at
CF. At Estrées-Mons (EM) and Norwich (NO) the two N treatments
were conducted in adjacent areas as randomised block designs in
which the 16 genotypes were tested in three replicates. Plot size
was 6 × 1.2 m at NO and 5 × 1.3 m at EM. In 2006–7 and 2007–8,
the experiments were sown on 4 and 10 October at SB, 18 and 19
 O. Gaju et al. / Field Crops Research 123 (2011) 139–152
Table 1
N fertilizer amount applied in high N (HN) and low N (LN) treatment, soil type, soil mineral N (SMN) to 90 cm soil depth in February, plant available water at field capacity
to 120 cm and soil organic matter percentage at Clermont-Ferrand (CF), Estrées-Mons (EM), Norwich (NO) and Sutton Bonington (SB) in 2006–7 and 2007–8.
Sites CF EM
N treatment HN LN HN
2006–7
N fertilizer kg N ha−1 240 40 240
Soil type Clay Fine clay loam
SMN kg N ha−1 73.4 43.0
Plant available soil water (mm) 129 306
Soil organic matter (%) 2.9 1.9
2007–8
N fertilizer kg N ha−1 240 40 210
Soil type Clay loam Fine clay loam
SMN kg N ha−1 62.1 54.0
Plant available soil water (mm) 122 306
Soil organic matter (%) 3.1 2.0
October at NO, 17 and 22 October at EM and 6 and 11 November
at CF, respectively. Across experiments different seed rates were
used sufficient to establish a target of 200 plants m−2 in the spring
and ranged from 250 to 350 seeds m−2 . The previous crop at CF,
EM, NO and SB was sunflower, winter oats, winter oilseed rape
and winter oilseed rape in 2006–7 and winter barley, winter oats,
sugar beet and winter oilseed rape in 2007–8, respectively. The
high N (HN) fertilizer treatment was intended to replicate com-
mercial practice and was calculated using methods described in
Anon (2000) in the UK and Rémy and Hébert (1977) in France using
measurements of the amount of mineral N in the soil in February.
Each HN treatment was applied using either three (at SB and NO)
or four (at EM and CF) split applications. In each experiment an
initial application of 34–60 kg N ha−1 was applied in March. In the
experiments at SB and NO, the remainder was applied split approx-
imately two-thirds at the start of stem extension (growth stage (GS)
31; Zadoks et al., 1974) and one-third at flag leaf emergence (GS39).
In the experiments at EM and CF the remainder was applied split
approximately two-fifths at GS31, two-fifths at GS39 and one-fifth
at anthesis (GS61). In the low N (LN) treatment, the amount of N
applied varied in the experiments from 0 to 50 kg N ha−1 , depend-
ing on soil mineral N in February, and was applied at GS31. The exact
amount of N applied under LN was adjusted in each site-season
according to the soil mineral N measured in February with the aim
of providing ca. 100 kg N ha−1 from the combined soil mineral N
and fertilizer N corresponding to a moderate to severe N limitation
sufficient to reduce grain yield by ca. 30% compared to HN condi-
tions. In seven of the eight site-seasons, the combined soil mineral N
and fertilizer N amount was thus in the range 65.0–113.4 kg N ha−1 .
In the remaining site-season (NO08), the combined soil and fertil-
izer N was slightly lower at 45.9 kg N ha−1 . In this site-season, the
soil mineral N in February was 45.9 kg N ha−1 , but was not sup-
plemented with applied fertilizer N since soil mineral N results
for this experiment were not available until after fertilizer deci-
sions were made. The total amount of N applied in the LN and HN
treatments in each experiment is given in Table 1. All N fertilizer
was applied as granules of ammonium nitrate (34.5% N) except
for the first three applications in EM that were applied as a liq-
uid solution (15% ureic acid, 7.5% ammonium, 7.5% nitrate). Each
split was applied on the same calendar date per genotype. All other
crop inputs including weed, disease and pest control, and potas-
sium, phosphate and sulphur fertilizers, were applied at levels to
prevent non-N nutrients or weeds, diseases and pests from lim-
iting yield. Plant growth regulator was applied as chlormequat at
onset of stem extension (GS31) in the experiments at EM, NO and
SB.
The 16 genotypes were selected as UK and French-bred culti-
vars/advanced breeding lines to represent feed wheat (Alchemy,
Arche, Beaver, Consort, Paragon, Rialto, Robigus and Savannah) and
141
NO SB
LN HN LN HN LN
50 180 20 210 0
Sandy clay Clay loam
50.6 96.2
113 165
2.2 3.0
50 200 0 210 30
Clay loam Sandy loam
45.9 35.0
123 70
1.9 1.6
bread-making wheat (CF9107, CF99102, Perfector, Quebon, Recital,
Renan, Soissons and Toisondor) types. They were shown to be con-
trasting for NUE and its components (LeGouis et al., 2000; Foulkes
et al., 2006) or contrasting for their grain protein concentration
(GPC) at a given yield (i.e. significant grain protein deviation (GPD)
from the overall negative grain yield-protein concentration rela-
tionship; Oury et al., 2003). CF9107 and CF99102 are advanced
INRA breeding lines. For simplicity, these two advanced breeding
lines are referred to hereafter as cultivars in the text. The classifica-
tions of the cultivars according to allelic expression of the Ppd-D1
gene for photoperiod insensitivity and for the Rht-D1 and Rht-B1
semi-dwarf genes for plant height is given in Table 2.
2.2. Crop measurements
Anthesis date (AD) was recorded when 50% of the ears carried
visible stamens (GS61) in all plots in each experiment. Crop dry
mass and N content were assessed at GS61 in all plots in seven
experiments (not assessed at NO in 2006–7). All cultivars were
sampled on the date of reaching GS61, i.e. different cultivars were
sampled on different calendar dates. At CF and SB, plant material
was removed from a 0.64 m2 area from each plot by cutting the
shoots at soil level. On a 10% sub-sample (by FW) the weights of the
straw and ears were recorded separately after drying at 80 ◦ C for
48 h. At EM and NO, on a random sample of 50 ear-bearing shoots
removed from each plot by cutting at ground level, the weights
of the straw and ears at GS61 were recorded separately after dry-
ing at 80 ◦ C for 48 h. At all four sites, the concentration of N in the
straw and ears was measured using the Dumas method. Nitrogen
Nutrition Index (NNI) was estimated according to the ratio of the
actual above-ground crop N% at anthesis and the critical N% (N%ct ),
where N%ct was estimated according to the ‘critical dilution curve’
described by Justes et al. (1994). At CF and SB, the above-ground N
and DM per unit ground surface area at GS61 was estimated directly
from the measurements taken on the quadrat sample. At EM and
NO, the above-ground DM and N per unit ground surface area at
GS61 was estimated by multiplying the above-ground DM and N
per ear-bearing shoot at GS61 by the number of ear per m2 esti-
mated at harvest. Previous studies have shown that for wheat the
number of ears m−2 changes little between anthesis and harvest
under either low N or high N conditions. For example, Pask (2009)
in a recent field study with experiments in the UK and New Zealand
showed that in each environment there was no statistically sig-
nificant change in ears m−2 between anthesis and harvest under
either low N or high N conditions. In all plots in all experiments,
crop height was measured ca. 14 d after the end of anthesis (GS69)
by measuring the height to the top of the ears at three locations in
each plot.
142 O. Gaju et al. / Field Crops Research 123 (2011) 139–152

Table 2
Reduced height Rht-B1a/Rht-B1b, Rht-D1a/Rht-D1b, and photoperiod insensitivity Ppd-D1a/Ppd-D1b classes, and average flowering dates and plant height of 16 cultivars.
Values represent means across high and low N treatments in eight site-seasons (Clermont Ferrand, Estrées-Mons, Norwich and Sutton Bonington in 2006–7 and 2007–8).
The Rht-B1b and Rht-D1b alleles confer semi-dwarf stature and the Ppd-D1a allele confers insensitivity to photoperiod.

Genotypes Ppd-D1a/Ppd-D1b Rht-B1a/Rht-B1b Rht-D1b/Rht-D1a Flowering date Plant height (cm)

Alchemy Ppd-D1b Rht-B1a Rht-D1b 4 June 74.6

Arche Ppd-D1b Rht-B1a Rht-D1a 28 May 76.9
Beaver Ppd-D1b Rht-B1a Rht-D1b 4 June 66.9
CF9107 Ppd-D1b Rht-B1a Rht-D1b 25 May 71.3
CF99102 Ppd-D1b Rht-B1b Rht-D1a 27 May 83.6
Consort Ppd-D1b Rht-B1a Rht-D1b 3 June 70.1
Paragon Ppd-D1b Rht-B1a Rht-D1a 1 June 91.9
Perfector Ppd-D1b Rht-B1a Rht-D1b 31 May 72.4
Quebon Ppd-D1b Rht-B1a Rht-D1b 31 May 78.3
Recital Ppd-D1a Rht-B1b Rht-D1a 19 May 67.4
Renan Ppd-D1b Rht-B1b Rht-D1a 26 May 76.5
Rialto Ppd-D1b Rht-B1a Rht-D1b 1 June 74.0
Robigus Ppd-D1b Rht-B1b Rht-D1a 1 June 72.3
Savannah Ppd-D1b Rht-B1a Rht-D1b 2 June 73.7
Soissons Ppd-D1a Rht-B1b Rht-D1a 21 May 73.3
Toisondor Ppd-D1a Rht-B1a Rht-D1b 26 May 62.6

In all plots in all experiments, a small plot combine was used to
determine the grain yield per unit of ground surface area (GY) from
an individual plot area of at least 5 m2 , and values adjusted to 0%
moisture at harvest (GS92). Thousand grain weight was measured
on a representative grain sample of 20 g. The grain was counted on a
Numigral (Villeneuve-La-Garenne, France) or a Contador (Pfeuffer,
Germany) seed counter and the weight corrected to 0% moisture.
Before machine harvesting, in all plots of all experiments crop
dry mass and N content were assessed on a random sample of
ca.100 ear-bearing shoots removed from each plot by cutting at
ground level. The number of shoots in each sample was counted.
The weights of the straw, and chaff and grain (after threshing the
ears) were recorded, after drying at 80 ◦ C for 48 h. The concentration
of N in the straw, chaff and grain was measured using the Dumas
method. Using these data, estimates of grain number per unit of
ground surface area (GN), ear number per unit of ground surface
area, grain number per ear and DM of straw, chaff and grain per unit
of ground surface area were calculated. The harvest index (HI) and
N harvest index (NHI) were calculated as the proportion of DM and
N in the above-ground plant which is in the grain at harvest, respec-
tively. The nitrogen-use efficiency (NUE) was calculated by dividing
the machine-harvested DM grain yield (kg DM ha−1 , 0% moisture
content) by the amount of N available to the crop from the soil and
fertilizer (kg N ha−1 ). The amount of N available to the crop from the
soil was taken as the amount of soil mineral N measured in Febru-
ary (Table 1). It is recognised that some mineralization of organic N
may occur post-February during the season resulting in an overes-
timation of NUE, but it is considered this amount of additional soil
mineral N would be similar for each cultivar and would not mean-
ingfully affect differences in NUE or NUpE amongst the cultivars
or the N × cultivar interaction reported. A similar assumption was
made in many previous studies, e.g. investigations by LeGouis et al.
(2000), Barraclough et al. (2010) and Berry et al. (2010).
The N-uptake efficiency was calculated by dividing the above-
ground N at harvest (kg N ha−1 ) by the amount of N available to the
crop from the soil and fertilizer (kg N ha−1 ); and the N-utilization
efficiency was calculated by dividing the machine-harvested DM
grain yield (kg DM ha−1 , 0% moisture content) by the above-ground
N at harvest (kg N ha−1 ). The crop N Remobilization Efficiency (NRE)
was calculated as:
AGNH − (AGNH − GN)
(1)
AGNA
where AGNA is the above-ground N at anthesis (kg N ha−1 ), AGNH
(kg N ha−1 ) is the above-ground N at harvest and GN (kg N ha−1 ) is
the grain N at harvest (kg N ha−1 ).
Lodging was defined as the stems leaning at an angle of at least
45◦ from the vertical. Close to the time of harvest the proportion of
the plot area that was lodged was visually assessed to the nearest
0.05.
2.2.1. Soil mineral sampling
Soil mineral N (0–120 cm in EM, 0–90 cm soil depth at other
sites) was assessed in early February to quantify soil N supply.
Samples were taken from Rialto plots in the LN treatment in three
replicates. Six cores were taken per sampled plot and separated
into 0–30, 30–60, 60–90 cm and 90–120 cm horizons. Samples were
bulked per horizon to give 3/4 samples per plot, one for each
soil horizon. The samples were analysed for soil mineral N (NO3 −
and NH4 + ). The soil N analysis was carried out by shaking 40 g
of soil sample in 2 M KCl; the extract solution after sieving was
analysed for nitrate-N and ammonium-N. The concentrations of
NO3 − were determined with the colorimetric cadmium reduction
method (Wagner, 1974), and of NH4 + with a spectrophotometric
method by which a coloured complex of NH4 + and sodium salicylate
is formed in the presence of prussidic acid and chloride (Benesch
and Mangelsdorf, 1972).
2.2.2. Senescence parameters
Senescence kinetics of the flag leaf of five main stems at CF or of
the whole canopy at other sites were assessed visually by recording
the percentage green area senesced using a standard diagnostic key
based on a scale of 0–10 (100% senesced). The same diagnostic key
was used at all sites, and at each site one operator assessed senes-
cence scores throughout a given season. Assessment was carried
out twice weekly after anthesis until full canopy senescence. The
data were then fitted against thermal time from anthesis (GS61;
base temperature of 0 ◦ C) using a modified version of an equation
with five parameters consisting of a monomolecular and a logistic
function (Eq. (2); Génard et al., 1999).
 
score = p0 + p1 × 1 − e(−p2×TTA/p1)
10 − p1 − p0
+ (2)
(−4×p3×((TTA−p4)/(10−p1−p0)))
1+e
where p0 is the initial senescence score at GS61; p1 is the senes-
cence score of first phase, p2 is the maximum rate of first phase; p3
is the maximum rate of second phase; p4 is the time when rate is
maximum in second phase; TTA is the thermal time post-anthesis.
This function describes senescence as a two-phase process with
a slow and a rapid phase (Fig. 1). Fitted kinetics were obtained by
minimizing the residual sum of square between observed and fitted
 O. Gaju et al. / Field Crops Research 123 (2011) 139–152 143

12
Slow phase senescence Rapid phase senescence

10
Leaf senescence score

p3 P4
6
SENONSET

p1 p4
2 P1

p0 P2
0 p2

0 200 400 600 800 1000

Thermal time post anthesis (oCd)

Fig. 1. Schematic representation of the senescence kinetics for the monomolecular
and a logistic function of Génard et al. (1999) showing the slow and rapid phases
of post-anthesis senescence. Senescence was scored visually from zero (leaves fully
green) to 10 (leaves fully senesced) from anthesis to complete canopy senescence.
Observed scores were fitted against accumulated thermal time after anthesis. Equa-
tion parameters are: p0, senescence score at anthesis; p1, senescence score at the
end of the slow phase; p2, maximal rate of senescence during the slow phase; p3
maximum rate of senescence during the rapid phase; and p4, thermal time when
p3 is reached. The onset of the senescing phase (SENONSET ) is taken as at the thermal
time at the end of the slow phase, and the rate of the senescence phase (SENRATE ) is
taken as p3.
data. The duration of each phase and the total duration of the senes-
cence period were derived after the inflexion point separating the
two phases had been determined as the thermal time when the sec-
ond derivative of the equation reached its maximum. The duration
of the first phase of senescence, in which green area loss is slow,
corresponds to the onset of senescence (SENONSET ; ◦ C days) and the
duration of the second phase, in which most of the green area loss
occurs, is the duration of the rapid senescing phase (SEN; ◦ C days).
In the present study, the analysis focuses on the rapid phase of post-
anthesis senescence, and the onset of post-anthesis senescence is
defined as SENONSET and the rate of post-anthesis senescence is
defined as the rate of the rapid phase of senescence (SENRATE ), i.e.
p3 in Eq. (2).
2.3. Statistical analysis
Analysis of variance with the GLM procedure of SAS statistical
software (SAS 9th edition, SAS Institute Inc., Cary, NC, USA) for a
split-plot design (CF and SB) or a fully randomised plot design (EM,
NO) were used to test for differences between the crop variables at
individual sites (SAS, 9th edition). For the combined ANOVA of the
data set from all eight experiments ANOVA (SAS 9th edition) was
performed using the following model:
Ygtek =  + ˛g + nt + se + ˛ngt + ˛sge + ˛nsgte + r(ns)tek + εgtek
where Ygtek is the trait for genotype g grown in block k in site-
season e and N treatment t,  is the general mean, ˛g is the genotype
main effect, nt is the N treatment main effect, se is the site-season
main effect, ˛ngt is the genotype × N treatment interaction, ˛sge is
the genotype × site-season interaction, ˛nsgte is the genotype × N
treatment × site-season interaction, r(ns)tek is the effect of block in
N treatment × site-season, and εgek is the residual effect.
An unbalanced design ANOVA procedure (SAS 9th edition) was
used to assess the effect of the Ppd-D1a/Ppd-D1b alleles for pho-
toperiod insensitivity/sensitivity on crop variables and interactions
with N treatments and site-seasons. Ordinary least square linear
regression was used to assess whether two crop variables were
Fig. 2. Machine-harvested grain yield (0% moisture content) of 16 cultivars (Arc,
Arche; Alc, Alchemy; Bea, Beaver; CF91, CF9107; CF99, CF99102; Con, Consort;
Que, Quebon; Par, Paragon; Per, Perfector; Rec, Recital; Ria, Rialto; Ren, Renan;
Rob, Robigus; Sav, Savannah; Soi, Soissons; Toi; Toisondor) under high (HN) and
low N (LN) treatments. Values represent means across 8 site-seasons at four
sites (Clermont-Ferrand, CF; Estrées-Mons, EM; Norwich, NO; Sutton Bonington,
SB) in 2006–7 and 2007–8. (DF = 497, SED (Genotype) = 0.08***, SED (N treat-
ment × genotype) = 0.12***).
correlated amongst the 16 cultivars. Linear regression with groups
was used to test whether the relationship between two variables
amongst the 16 cultivars differed between experiments. Genstat
8.1 (www.genstat.com) was used for all regression analyses.
3. Results
3.1. Grain yield, yield components and anthesis date
Averaged across all cultivars, the reduction in GY in the LN com-
pared to the HN treatment ranged from 0.36 t ha−1 (6%) at EM07
to 4.97 t ha−1 (55%) at NO08 (P < 0.001; Table 3), with an average
reduction across all eight experiments of 2.22 t ha−1 (29%; P < 0.001;
Fig. 2). The largest GY loss occurred at NO08 where the site had
a large yield potential and a low soil mineral N availability. The
interaction between N treatment and cultivar for the individual
experiments was statistically significant in NO07 and CF08. The
combined ANOVA for the eight experiments showed an interaction
between N treatment and cultivar (P < 0.05; Fig. 2). On average the
proportional loss in GY under LN conditions was less for CF9107
(0.25), Arche, Consort and Robigus (0.26) than for Rialto (0.32)
and Quebon (0.33). There was a significant site-season × N treat-
ment × cultivar effect (P < 0.05, Table 3). The high tolerance of N
limitation for Consort was mainly observed in experiments at NO
and to a lesser extent at EM, and the high tolerance of CF9107
mainly at SB. The low tolerance of N limitation of Rialto was mainly
found in experiments at the NO site. The high tolerance of Arche
and the low tolerance of Quebon of N limitation were generally
detected across all experiments. The highest absolute GY in both
the HN and the LN treatment was achieved by Alchemy and the
lowest in both cases by Renan; and overall there was a positive lin-
ear relationship amongst cultivars between GY under HN and LN
conditions (R2 = 0.84). Nevertheless, some cultivars changed rank-
ings. For example, Rialto dropping from 4th highest under HN to
9th highest under LN conditions; Consort and Robigus also changed
rankings, ranking relatively higher under LN than under HN.
Turning to consider yield components, overall the main effect
of N supply was on GN, with a reduction from 18,512 to 13,244
grains m−2 (−28.1%; P < 0.001; Table 3), rather than individual grain
dry mass which was not modified (P = 0.07) by the N treatment
144 O. Gaju et al. / Field Crops Research 123 (2011) 139–152

Table 3
Combine grain yield (0% DM), above-ground nitrogen at harvest (AGN), nitrogen harvest index (NHI), grain N concentration (GNC), grains m−2 and harvest index (HI) in
high N (HN) and low N (LN) treatments at four sites (Clermont-Ferrand, CF; Estrées-Mons, EM; Sutton Bonington, SB; Norwich, NO) in 2006–7 (07) and 2007–8 (08). Values
represent means of 16 cultivars.

Yield (t DM ha−1 ) Above-ground N Nitrogen harvest GNC (%) Grains (m−2 ) Harvest index
(kg N ha−1 ) index

HN LN GxN HN LN GxN HN LN GxN HN LN GxN HN LN GxN HN LN GxN

***
CF07 5.49 4.29 Ns 182.6 93.2 ns 0.73 0.72 ns 2.44 1.56 15,238 11,787 ns 0.44 0.45 ns
*** * **
CF08 7.44 5.38 236.7 116.5 0.71 0.78 ns 2.25 1.71 ns 20,151 14,339 0.44 0.43 ns
EM07 6.48 6.12 Ns 223.0 130.8 ns 0.71 0.80 ns 2.47 1.69 ns 17,501 15,865 ns 0.47 0.48 ns
* *
EM08 8.44 6.36 Ns 239.0 118.9 ns 0.82 0.85 ns 2.33 1.61 ns 19,429 14,671 0.52 0.49
*** ** *** ** *** ***
NO07 6.62 3.87 195.6 74.5 0.84 0.89 2.49 1.73 18,094 9,658 0.52 0.52
*** *
NO08 8.98 4.01 Ns 204.1 58.7 ns 0.89 0.90 ns 2.06 1.31 18,266 8,738 0.60 0.59 ns
SB07 8.24 6.73 Ns 247.4 150.1 ns 0.66 0.68 ns 1.84 1.48 ns 20,688 16,310 ns 0.47 0.45 ns
* *
SB08 9.65 6.86 Ns 289.5 127.1 ns 0.76 0.79 ns 2.13 1.40 18,726 13,971 ns 0.50 0.53

DF 385 377 373 386 376 375

LSDa (Genotype) 0.15 5.99 0.01 0.04 402 0.02
LSD (N treatment) 0.05 2.12 0.001 0.01 142 0.01
LSD (G × N) 0.20 8.47 0.02 0.06 569 0.02
LSD (Site × N) 0.10 4.23 0.01 0.03 284 0.01
LSD (Year × Site × G × N) 0.57 23.9 0.04 0.16 1609 0.06
a
Least significant difference (˛ = 0.05).
*
Significance at the 5% (P = 0.05) level.
**
Significance at the 1% (P = 0.01) level.
***
Significance at the 0.1% (P = 0.001) level.

(data not shown). The N treatment × cultivar interaction was sig-

nificant for both yield components (P < 0.05). Grain number per unit
of ground surface area was maintained relatively better under LN 40
conditions for Robigus, Arche and Soissons (−26%) than for Quebon a
NUE (kg grain DM kg-1 N available)

(−34%) and Renan (−32%). Although the main effect of N treat- 35

ment was not significant for grain dry mass, there was a large range
of responses amongst cultivars to increasing N supply from −3.57
30
to +4.60 mg DM grain−1 . These responses partly explained the GY
responses to N treatment, e.g. Rialto (−1.61 mg DM grain−1 ) and
25
Quebon (−3.57 mg DM grain−1 ) compared to Robigus (+1.80 mg DM
grain−1 ) and Consort (+1.42 mg DM grain−1 ).
20
Averaging across experiments and cultivars, the effect of N treat-
ment on anthesis date was not statistically significant (P = 0.27, data
15
not shown). Overall, AD ranged from 19th May (Recital) to 4th June
(Beaver; Table 2). Averaging across the eight experiments, there
10
was a positive linear relationship amongst cultivars between AD
0.50 0.70 0.90 1.10 1.30
and GY under HN (R2 = 0.53, P < 0.01) and LN conditions (R2 = 0.44,
P < 0.01). There was a positive association at each of three sites N Uptake Eff. (kg AGN kg -1 N available)
(CF, SB and NO), and parallel regression analysis showed that the
slopes at these sites differed; with GY increasing by 0.16, 0.29 105
and 1.21 kg DM ha−1 for every day’s delay in AD at NO, CF and b
NUE (kg grain DM kg-1 N available)

95
SB, respectively (data not shown). Three of the French cultivars
(Soissons, Recital and Toisondor) were photoperiod insensitive 85
(Ppd-D1a) and the remaining 13 cultivars were photoperiod sen-
75
sitive (Ppd-D1b; Table 2). Analysis of variance including Ppd-D1 as
a factor showed that, averaging across site-seasons and N treat- 65
ments, the Ppd-D1a cultivars flowered 9 days earlier and yielded
55
0.38 t ha−1 less than the Ppd-D1b cultivars (P < 0.001; Table 4). With
regard to yield components, the Ppd-D1a cultivars showed a trend 45
to increase GN by 405 grains m−2 (+2.6%; P = 0.07), but decrease
35
grain weight by 3.34 mg DM grain−1 (−7.9%) compared to the Ppd-
D1b cultivars (P < 0.001; Table 4). Neither the N treatment × Ppd-D1 25
allelic class nor the site-season × N treatment × Ppd-D1 allelic 0.50 1.00 1.50 2.00
class interaction was statistically significant for GY or yield
N Uptake Eff. (kg AGN kg-1 N available)
components.
Fig. 3. Linear regression of N-uptake efficiency (kg above-ground N (AGN) at har-
vest per kg N available from soil and fertilizer N) on N-use efficiency (kg grain DM
3.2. Nitrogen-use efficiency and NUE components per per kg N available from soil and fertilizer N) amongst 16 cultivars in (a) high N
treatment: (♦) Clermont Ferrand R2 = 0.01, (×) Estrées-Mons R2 = 0.05; () Norwich
R2 = 0.01 and () Sutton Bonington R2 = 0.01 and (b) low N treatment: (♦) Cler-
In each experiment, NUE was increased in the LN compared mont Ferrand R2 = 0.01, (×) Estrées-Mons R2 = 0.03; () Norwich (y = 56.15x + 6.00,
with the HN treatment (P < 0.01, Fig. 3), with on average values R2 = 0.40, P < 0.01) and () Sutton Bonington R2 = 0.17. Values represent means across
increasing from 28.6 (range 18.2–38.1 kg DM kg−1 N) to 67.0 (range 2006–7 and 2007–8.
 O. Gaju et al. / Field Crops Research 123 (2011) 139–152 145

Table 4
Machine-harvested grain yield (0% moisture content; GY), N-utilization efficiency (kg grain DM per kg above-ground N at harvest, NUtE), grain protein concentration (GPC),
N harvest index (NHI), harvest index (HI), above-ground N at anthesis (AGNA ), post anthesis N uptake (PANU), above-ground N at harvest (AGNH ), grains per m2 , thousand
grain weight (TGW) and biomass production efficiency (kg above-ground DM per kg above-ground N at harvest; BPE) in high N (HN) and low N (LN) treatments at four sites
(Clermont Ferrand, CF; Estrées-Mons, EM; Norwich, NO; Sutton Bonington, SB) in 2006–7 and 2007–8. Values represent means for Ppd-D1a (3 cultivars) and Ppd-D1b (13
cultivars) groups across eight site-seasons; except for AGNA , PANU and AGNH across seven site-seasons (all site-seasons except NO 2006–7).

GY (t ha−1 ) NUtE GPC (%) NHI HI AGNA PANU AGNH Grains m−2 TGW (g) BPE
(kg DM kg N−1 ) (kg N ha−1 ) (kg N ha−1 ) (kg N ha−1 ) (kg DM kg−1 N)

HN Ppd-D1a 7.34 33.7 12.8 0.76 0.52 167.8 56.3 224.1 18871 39.3 65.8
HN ppp-D1b 7.74 34.1 12.8 0.77 0.49 187.8 43.2 231.4 18506 42.1 69.7
LN Ppd-D1a 5.15 50.7 9.1 0.80 0.51 86.9 24.6 111.5 13524 38.7 100.5
LN Ppd-D1b 5.52 52.2 8.9 0.80 0.49 92.9 23.1 115.9 13079 42.6 107.1
DF 721 707 722 721 711 630 610 619 710 713 705
SED (Ppd-D1) 0.07 0.53 0.11 0.003 0.004 2.54 2.90 1.92 207 0.34 1.14
SED (N x Ppd-D1) 0.12 0.95 0.21 0.01 0.008 4.61 5.23 3.47 374 0.61 2.05

40.7–100.7 kg DM kg−1 N) kg DM kg−1 N, respectively. The cultivars
differed in NUE in each site-season (P < 0.001); and there was an
interaction between N treatment and cultivar at NO07 and CF08
(P < 0.05). These interactions reflected those observed for GY since
the N fertilizer amount was the same for each cultivar within a
given N treatment in a given experiment.
3.2.1. Nitrogen-uptake efficiency
NUpE increased from 0.84 under HN to 1.29 under LN condi-
tions (Table 5). There were differences amongst the sites in NUpE
(Fig. 3); for example, overall NupE under LN was lowest at CF (0.98),
intermediate at NO and EM (1.16 and 1.27, respectively) and high-
est at SB (1.76). This indicated that more soil N mineralization
post-February occurred at SB than at other sites. The overall N treat-
ment × cultivar interaction was not statistically significant. For the
mean values across years, the linear relationship between NUpE
and NUE amongst the cultivars is shown at each site under HN and
LN conditions in Fig. 3. In only one of the site/N treatment com-
binations was there a statistically significant relationship: at NO
(R2 = 0.40, P < 0.01) under LN conditions, although there was also a
trend at SB for a positive relationship under LN (R2 = 0.17; P = 0.10).
Therefore, overall the NUpE effect explained only a small amount
of the phenotypic variation in NUE amongst cultivars in the experi-
ments. Averaging values across the four sites, the linear regression
of NUpE on NUE amongst cultivars was not statistically signifi-
cant under either HN or LN conditions. For individual experiments,
the N × cultivar interaction was not significant except for CF08.
Although the overall interaction was not significant there were
differences in NUpE amongst the cultivars in the range 1.14–1.35
under LN 479 and 0.74–0.92 under high N conditions. For example,
nder LN Robigus and Arche demonstrated higher NUpE than Rialto
and Quebon (P < 0.05). There was no significant correlation amongst
cultivars between either AD or plant height and NUpE under either
LN or HN conditions.
Overall above-ground N uptake at harvest was reduced from
227 kg N ha−1 under HN to 109 kg N ha−1 under LN conditions
(P < 0.001; Table 5). AGNH decreased in each experiment under
LN supply (P < 0.01), with decreases ranging from 89 (CF07) to
145 (NO08) kg N ha−1 (Table 3). The cultivar effect was signif-
cant in all experiments (P < 0.05), with an overall range of 200
(Recital) to 247 (Quebon) kg N ha−1 under HN and 97 (Recital) to
114 kg N ha−1 (CF99102) under LN conditions (P < 0.001; Table 5).
Although the N treatment × cultivar interaction was only signfi-
cant in two individual experiments (CF08 and NO07; P < 0.05 and
<0.001, respectively), there was overall a N treatment × cultivar
interaction (P < 0.001). Savannah maintained N uptake relatively
better under LN (113 kg N ha−1 ; 53% of HN treatment value) than
Paragon (104 kg N ha−1 ; 45% of HN treatment value) and Que-
bon (113 kg N ha−1 ; 46% of HN value). The three-way interaction
with site-season was not statistically significant. The photoperiod
insensitive cultivars acquired less N than the sensitive cultivars
under both LN (116 vs 111 kg N ha−1 ) and HN conditions (231 vs
224 N ha−1 ); the N treatment × Ppd-D1 allelic class interaction was
not statistically significant (Table 4).
Above-ground N uptake at anthesis was measured in seven of
the eight experiments (not measured at NO07). The main effects
of N (P < 0.001), cultivar (P < 0.01) and N × cultivar interaction were
signficant (P < 0.001). The three-way interaction with site-season
was again not significant. Averaging across these seven experi-
ments, under HN 184 kg N ha−1 was absorbed at anthesis compared
to 90 kg N ha−1 under LN conditions (Table 6), but under both N
treatments it represented 79% of AGNH . AGNA varied amongst the
cultivars from 151 (Recital) to 203 (Robigus) kg N ha−1 under HN
and from 79 (Recital) to 100 (Consort) kg N ha−1 under LN condi-
tions. As at harvest, Rialto showed a poor capacity for N uptake
under LN (measured as proportion of N uptake under HN condi-
tions = 0.45). CF99102 (0.53) and Recital (0.52) showed the best
relative N uptakes at anthesis. Savannah, which showed the best
capacity for N uptake under LN at harvest, showed poorer relative
N uptake at anthesis, with a similar value to Rialto (0.46). Later AD
in the photoperiod sensitive cultivars was associated with greater
AGNA in both N treatments; particularly so under HN conditions
(P < 0.001; Table 4).
Post-anthesis N uptake (PANU) was overall 47 kg N ha−1 under
HN and 24 N kg ha−1 under LN conditions (P < 0.001; Table 6).
The cultivars ranged from 26 (Robigus) to 64 (CF9107) kg N ha−1
under HN and 8 (Consort) to 36 (CF9107) kg N ha−1 under LN
(P < 0.001) conditions. PANU amongst the cultivars was negatively
associated with AGNA under both HN (R2 = 0.49, P < 0.01) and LN
(R2 = 0.39; P < 0.05). Anthesis date was positively associated with
AGNA (R2 = 0.49, P < 0.01) and PANU under HN conditions (R2 = 0.42,
P < 0.05). Whereas under LN, AD was not significantly associated
with AGNA or PANU. There was no overall effect of Ppd-D1 allelic
class on PANU, although there was a trend for an interaction with
N treatment (P = 0.06), with the Ppd-D1a and Ppd-D1b cultivars
acquiring similar amounts of AGNA under LN conditions, but the
Ppd-D1a cultivars acquiring 12 kg N ha−1 more N than the sensitive
cultivars under high N. The three-way interaction with site-season
was not statistically significant.
3.2.2. Nitrogen-utilization efficiency
Overall NUtE increased from 34.0 under HN to 52.1 kg DM kg−1 N
under LN conditions. Averaging across site-seasons, there was a
positive linear relationship between NUtE and NUE amongst the
cultivars accounting for 61% and 77% of the phenotypic variation
in NUE under LN and HN conditions, respectively. There was a
N treatment × cultivar interaction (P < 0.001); Arche and CF9107
increased NUtE under LN relatively more than Savannah. Positive
linear regressions of NUtE on NUE were found in all site-seasons
under both LN and HN conditions (P < 0.05; Fig. 4). Overall there
was a trend for a positive correlation between AD and NUtE under
both HN (r = 0.47, P = 0.07) and LN (r = 0.42, P = 0.09). However, the
146 O. Gaju et al. / Field Crops Research 123 (2011) 139–152

Table 5
N-utilization efficiency (kg grain DM per kg above-ground N at harvest; NUtE), N-uptake efficiency (kg above-ground N at harvest per kg N available; NUpE), above-ground
N at harvest (AGNH ), N harvest index (NHI) and biomass production efficiency (kg above-ground DM per kg above-ground N at harvest; BPE) at eight site-seasons (four sites
(Clermont-Ferrand, Estrées-Mons, Norwich, Sutton Bonington) in 2006–7 and 2007–8) for 16 cultivars in high N (HN) and low N (LN) treatments. Values represent means
across eight site-seasons.

NUtE (kg DM kg−1 N) NUpE (kg AGN kg−1 N) AGN harvest (kg N ha−1 ) NHI BPE (kg DM kg−1 N)

HN LN HN LN HN LN HN LN HN LN

Alchemy 38.0 58.9 0.82 1.28 222.2 106.5 0.76 0.80 76.7 116.4
Arche 35.8 55.6 0.83 1.31 223.0 110.7 0.77 0.81 73.9 112.3
Beaver 35.3 53.8 0.82 1.25 221.1 104.9 0.77 0.79 70.4 109.3
CF9107 29.9 47.9 0.86 1.33 231.9 111.9 0.80 0.83 62.3 97.1
CF99102 30.8 47.2 0.89 1.35 242.4 113.9 0.80 0.82 65.7 104.4
Consort 35.1 55.1 0.81 1.26 220.2 105.8 0.73 0.78 71.8 105.9
Paragon 32.9 52.9 0.86 1.25 231.0 104.0 0.77 0.82 72.5 115.4
Perfector 35.8 56.1 0.82 1.30 222.5 108.0 0.76 0.80 64.9 108.8
Quebon 31.5 47.9 0.92 1.34 246.7 112.7 0.77 0.80 64.9 101.9
Recital 34.4 49.6 0.74 1.14 200.3 97.4 0.77 0.81 64.6 97.2
Renan 27.3 41.6 0.89 1.34 240.1 113.5 0.75 0.79 59.8 95.0
Rialto 34.3 51.5 0.87 1.30 234.9 109.8 0.76 0.79 68.5 102.4
Robigus 36.6 56.4 0.83 1.33 224.8 110.7 0.75 0.80 76.4 113.0
Savannah 39.0 56.3 0.79 1.34 213.5 112.9 0.76 0.80 72.5 110.2
Soissons 31.6 47.8 0.88 1.33 237.4 112.0 0.76 0.79 63.6 97.1
Toisondor 35.1 54.6 0.84 1.26 225.9 105.4 0.76 0.80 69.5 107.1

Mean 34.0 52.1 0.84 1.29 227.4 108.7 0.77 0.80 68.6 105.8
DF 483 483 486 497 481
LSD (Genotype) 1.27 0.04 6.50 0.01 4.02
LSD (N treatment) 0.45 0.02 2.30 0.004 1.42
LSD (G × N) 1.80 0.07 9.20 0.02 5.68

effect of Ppd-D1 allelic class was not statistically significant, and
there was no N treatment × Ppd-D1 allelic class interaction for
NUtE.
Grain yield can be divided into the components of N uptake
(AGNH ) and NUtE according to:
AGNH × NHI
Grain yield =
GPC/(5.7 × 100)
where NHI is the nitrogen harvest index, and 5.7 is the factor to con-
vert grain N content to grain protein concentration. N-utilization
efficiency at harvest will be positively associated with NHI and/or
negatively associated with GPC according to Eq. (3).
In the present study, NHI overall increased from 0.77 under HN
to 0.80 under LN conditions (Table 5). Overall cultivars differed in
the range 0.73 (Consort) to 0.80 (CF99102 and CF9107) under HN
and 0.78 (Consort) to 0.83 (CF9107) under LN conditions (P < 0.001;
Table 5), but the reponse of the cultivars to N supply did not differ
significantly. Ppd-D1 allelic class did not have a significant effect
on NHI. Differences amongst cultivars in GPC broadly showed the
anticipated differences between the higher yield potential feed
(3) cultivars and the lower yield potential bread-making cultivars in
the range 11.2% (Savannah) to 15.6% (Renan) under HN and 7.9
(Alchemy) to 10.8% (Rialto) under LN (Fig. 5). The decrease in GPC
under LN differed amongst cultivars in the range 3.0–4.4 (P < 0.01);
cultivars with highest GPC under HN conditions tended to decrease
by most under LN. Neither the Ppd-D1 allelic effect or the N treat-
ment × Ppd-D1 allelic class interaction was statistically significant
for GPC.

Table 6
Above-ground N at anthesis (AGNA ), above-ground N at harvest (AGNH ), post-anthesis N uptake (PANU) and crop N remobilisation efficiency (NRE) at seven site-seasons (all
site-seasons except NO 2006–7) for 16 cultivars in high N (HN) and low N (LN) treatments. Values represent means across seven site-seasons (NO07 not assessed).

AGNA (kg N ha−1 ) AGNH (kg N ha−1 ) PANU (kg N ha−1 ) NRE

HN LN HN LN HN LN HN LN

Alchemy 183.9 87.5 226.1 110.6 42.2 23.1 0.69 0.71

Arche 187.6 92.3 228.2 116.0 39.9 27.5 0.68 0.72
Beaver 183.3 88.3 227.4 110.6 44.1 22.7 0.69 0.71
CF9107 169.8 81.4 233.6 116.9 63.8 35.5 0.71 0.75
CF99102 185.6 97.7 245.6 119.1 56.5 21.4 0.70 0.73
Consort 195.4 99.7 226.2 110.0 31.4 8.3 0.66 0.72
Paragon 181.0 90.6 233.8 107.7 53.6 17.1 0.65 0.75
Perfector 184.4 89.2 228.7 112.6 44.4 22.1 0.65 0.69
Quebon 197.9 89.1 251.8 118.4 54.2 29.3 0.67 0.70
Recital 151.1 79.0 208.8 104.0 57.9 24.3 0.63 0.70
Renan 187.1 93.8 241.3 118.5 55.1 24.7 0.64 0.71
Rialto 188.3 85.3 242.2 114.4 52.1 28.5 0.69 0.72
Robigus 202.7 94.7 229.4 115.6 25.9 21.5 0.66 0.68
Savannah 192.8 88.7 219.3 118.7 26.4 30.4 0.69 0.70
Soissons 179.6 91.7 239.9 116.0 58.4 24.3 0.65 0.73
Toisondor 179.3 87.7 228.3 109.3 51.5 22.9 0.66 0.72

Mean 184.4 89.8 231.9 113.6 47.3 24.0 0.67 0.71

DF 434 423 414 402
LSD (Genotype) 9.67 7.23 10.8 0.036
LSD (N treatment) 3.42 2.56 3.82 0.013
LSD (G*N) 13.6 10.2 15.3 0.051
 O. Gaju et al. / Field Crops Research 123 (2011) 139–152 147

40 17
a a
NUE (kg grain DM kg -1 available N)

16

Grain protein conc. (%)

35
15
14
30
13
25 12
11
20
10
9
15
15 20 25 30 35 40 45
20 25 30 35 40 45 50

N-Ulizaon Eff. (kg grain DM kg -1 AGN) NUE (kg grain DM kg-1 N available)

12
105
b
NUE (kg grain DM kg -1 N available)

b 11

Grain protein conc. (%)

95

85 10

75 9

65
55
45
35
30 40 50 60
N-Ulizaon Eff. (kg grain DM kg -1 AGN)
Fig. 4. Linear regression of N-utilization efficiency (kg grain DM per kg above-
ground N (AGN) at harvest) on N-use efficiency (kg grain DM per per kg N available
from soil and fertilizer N) amongst 16 cultivars in (a) high N treatment: (♦) Clermont
Ferrand y = 0.31x + 11.27, R2 = 0.47, P < 0.001; (×) Estrées Mons y = 0.45x + 13.46,
R2 = 0.48; P < 0.01; Norwich () y = 0.29x + 22.35; R2 = 0.26, P < 0.05 and () Sutton
Bonington y = 0.88x + 3.74; R2 = 0.88, P < 0.001 and (b) low N treatment: (♦) Cler-
mont Ferrand y = 0.34x + 29.2; R2 = 0.33, P < 0.05; (×) Estrées-Mons y = 0.59x + 33.25;
R2 = 0.44, P < 0.01; () Norwich y = 1.00x + 9.97; R2 = 0.47, P < 0.01; () Sutton Bon-
ington y = 1.55x + 10.55; R2 = 0.73, P < 0.001. Values represent means across 2006–7
and 2007–8.
Averaging across years, there was no statistically significant
phenotypic correlation between NHI and NUE or between NHI and
NutE amongst cultivars in any site under either LN or HN con-
ditions. Thus, the relatively small genetic variation observed in
NHI appeared to explain little if any of the variation observed in
NUtE amongst the cultivars under HN or LN conditions. In con-
trast, averaging over years, GPC was negatively correlated with both
NUE (Fig. 5) and NUtE (data not shown) amongst cultivars in each
site under both LN and HN conditions. Parallel regression analy-
sis showed that slopes were significantly different amongst site/N
treatment combinations (P < 0.001).
Overall in these experiments, there was a negative correlation
between NUpE and NUtE amongst cultivars under HN (r = −0.68,
P < 0.05) but not under LN conditions (r = −0.21). With regard to
performance at the four sites, amongst the cultivars there was a
negative relationship between NUpE and NUtE at each of the two
French sites under both HN and LN conditions (P < 0.05; Fig. 6). In
addition, at NO there was a negative relationship between NUpE
and NUtE under HN conditions (P < 0.01).
Cultivar differences were also observed for the biomass pro-
duction efficiency (above-ground DM at harvest/AGNH ; BPE);
averaging across N treatments, values ranged from 78 kg DM kg−1 N
(CF9107 and Renan) to 97 kg DM kg−1 N (for Alchemy and Robigus)
(Table 5). The cultivars did not respond differently to N supply for
BPE. The phenotypic correlation between BPE and NUtE amongst
cultivars was significant under both HN (r = 0.86, P < 0.001) and LN
8
7
6
35 45 55 65 75 85 95 105
70 80
NUE (kg grain DM kg-1 N available)
Fig. 5. Linear regression of N-use efficiency (kg grain DM per per kg N avail-
able from soil and fertilizer N; NUE) on percentage grain protein concentration
(GPC) amongst 16 cultivars in (a) high N treatment: (♦) Clermont-Ferrand
y = −0.78x + 29.71, R2 = 0.43, P < 0.01; (×) Estrées Mons y = −0.41x + 24.71, R2 = 0.42;
P < 0.01; () Norwich y = −0.30x + 23.20, R2 = 0.33, P < 0.05; and () Sutton Boning-
ton y = −0.26x + 19.75, R2 = 0.72, P < 0.001 (b) low N treatment: (♦) Clermont-Ferrand
y = −0.20x + 18.49, R2 = 0.34, P < 0.05; (×) Estrées Mons y = −0.14x + 17.99, R2 = 0.36;
P < 0.05; () Norwich = −0.07x + 13.45, R2 = 0.48, P < 0.01; and () Sutton Bonington
y = −0.09x + 16.33, R2 = 0.89, P < 0.001.
(r = 0.84, P < 0.001) conditions. Interestingly, the Ppd-D1b cultivars
showed greater BPE than the Ppd-D1a cultivars (P < 0.001; Table 4),
although the Ppd-D1a and Ppd-D1b cultivars responded similarly to
N supply.
3.3. Crop nitrogen remobilization efficiency
The post-anthesis N remobilization efficiency increased from
0.67 under LN to 0.71 under HN conditons (P < 0.001; Table 6). Aver-
aging across site-seasons and N treatments, there was a trend for
differences amongst cultivars in the range 0.66 (Recital) to 0.73
(CF9107) (P < 0.07). The N × cultivar effect was not significant, but
there was a site-season × N × cultivar interaction (P < 0.05). This
was mainly associated with the NRE increasing relatively more
under LN for Paragon compared to Robigus at CF08, whereas in
other site-seasons the cultivars generally responded similarly to N
supply.
3.4. Relationships between canopy senescence-related
parameters and yield and NUE components
In 2007–8 SENONSET and SENRATE in the post-anthesis period
were measured at all sites. Averaging across cultivars and sites,
SENONSET was earlier under LN (497 ◦ Cd after anthesis) than under
HN conditions (633 ◦ Cd after anthesis). Under HN conditions aver-
aging across cultivars, SENONSET ranged from 596 (SB) to 633 ◦ Cd
(CF) after anthesis and under LN conditions from 446 (SB) to 552 ◦ Cd
after anthesis (NO). Averaging across sites, under HN SENONSET
148 O. Gaju et al. / Field Crops Research 123 (2011) 139–152

55 105
a
N Ut. Eff. (kg grain DM kg -1 AGN )

N-Ut. Eff. (kg grain DM kg-1 AGN)

50 95

45 85

40 75

35 65

30 55

25 45

20 35
0.50 0.70 0.90 1.10 1.30 200 400 600 800

N Uptake Eff. (kg AGN kg -1 N available) Onset of post-anthesis senescence ( oCd)

80 9
b b
N Ut. Eff. (kg grain DM kg -1 AGN )

75 8
70

Grain yield (t ha -1)

7
65
60 6

55 5
50
4
45
3
40
35 2
0.50 1.00 1.50 2.00 200 400 600 800
Onset of post-anthesis senescence ( oCd)
N Uptake Eff. (kg AGN kg-1 N available)
Fig. 7. Linear regression amongst 16 cultivars in low N treatment of onset of post-
Fig. 6. Linear regression of N-uptake efficiency (kg above-ground N(AGN) at harvest
anthesis senescence (SENONSET ) in thermal time (base temp. 0 ◦ C) after anthesis
per kg N available from soil and fertilizer N) on N-utilization efficiency (kg grain
(GS61) on (a) N-utilization efficiency (kg grain DM per kg above-ground N (AGN)
DM per kg above-ground N (AGN) at harvest) amongst 16 cultivars in (a) high N
at harvest); (♦) Clermont-Ferrand R2 = 0.09; (×) Estrées Mons y = 0.08x + 13.06;
treatment: (♦) Clermont Ferrand y = −49.2x + 64.3, R2 = 0.52, P < 0.001; (×) Estrées
R2 = 0.32; P < 0.05; () Norwich y = 0.08 + 24.27; R2 = 0.50, P < 0.001; () Sutton
Mons y = −27.9x + 55.6, R2 = 0.32; P < 0.05; Norwich () y = −44.2x + 77.5 R2 = 0.62,
Bonington y = 0.05x + 31.14, R2 = 0.70, P < 0.01 and (b) grain yield (0% moisture con-
P < 0.001, and () Sutton Bonington R2 = 0.09, (b) low N treatment: (♦) Clermont-
tent): (♦) Clermont-Ferrand y = 0.002x + 4.01; R2 = 0.25, P = 0.05; (×) Estrées-Mons
Ferrand y = −41.8x + 87.5, R2 = 0.54, P < 0.001; (×) Estrées Mons y = −30.5 x + 89.8,
y = 0.01x + 1.56, R2 = 0.55; P < 0.01; () Norwich R2 = 0.11; () Sutton Bonington
R2 = 0.40; P < 0.01; Norwich () R2 = 0.01, and () Sutton Bonington R2 = 0.01.
y = 0.01x + 4.58, R2 = 0.38, P < 0.05. Values represent means in 2007–8.

ranged from 584 (Perfector) to 676 ◦ Cd after anthesis (Robigus)

and under LN from 382 (CF9107) to 580 ◦ Cd after anthesis (Savan- under LN (r = −0.59, P < 0.05). Although SENRATE showed a positive
nah). A correlation matrix showing the associations amongst the correlation with NUtE under LN conditions, this likely represented
16 cultivars between SENONSET , SENRATE , PANU, NUtE, NRE and an autocorrelation since SENONSET and SENRATE were positively cor-
GY is shown in Table 7 for both LN and HN conditions, based on related, i.e. cultivars which senesced later subsequently senesced
average performance across the four sites. Under LN conditions, at a faster rate. Under HN conditions, the correlations between
SENONSET was positively correlated with GY and NUtE (r = 0.80 and senescence parameters and each of GY, NutE and NRE were not sta-
0.84, respectively, P < 0.001; Fig. 7) and negatively correlated with tistically significant (Table 7). PANU was positively correlated with
NRE (r = −0.52, P < 0.05). NRE was also negatively correlated with GY GY (r = 0.52, P < 0.05) under LN conditions, but showed a neutral
relationship with GY under HN conditions. SENONSET was nega-
tively correlated with PANU under HN (r = −0.49, P < 0.05) but no
Table 7
Correlation coefficient between grain yield (t DM ha−1 , 0% moisture; GY), N- association was found under LN (Table 7). With regard to the indi-
utilization efficiency (kg grain DM per kg above-ground N at harvest; NUtE), vidual sites, under LN conditions, there was a positive association
post-anthesis N uptake (PANU; kg ha−1 ), rate of post-anthesis senescence (◦ Cd−1 ; between SENONSET and NUtE at EM, NO and SB (P < 0.05; Fig. 7a). The
SENRATE ); onset of post-anthesis senescence (◦ Cd after GS61; SENONSET ) and crop N overall negative association between NRE and SENONSET under LN
remobilisation efficiency (NRE) amongst 16 wheat cultivars in high N and low N
(P < 0.05) was signficant only at CF (r = −0.49; P < 0.05). Under HN
(highlighted in grey) treatments. Mean values for each cultivar over four sites in
2007–8 were used. conditions, the regressions between senescence parameters and
either GY, NUtE or NRE were not statistically significant in any of
GY NUtE PANU SENRATE SENONSET NRE
the sites.
GY *
0.87 ***
0.52*
0.73 ***
0.80 ***
−0.59*
NutE 0.85*** *
0.40 0.73*** 0.84*** −0.47
PANU −0.29 −0.60** *
0.53* 0.18 −0.51 4. Discussion
SENRATE 0.43 0.21 0.10 *
0.64** −0.47
SENONSET −0.06 0.33 −0.49* 0.01 *
−0.52*
NRE 0.13 −0.03 −0.06 0.02 −0.26 * These results allow us to discuss firstly the physiological basis
*
of genetic variation in NUE and responses to N supply and secondly
Significance at the 5% level (DF = 15).
**
Significance at the 1% level (DF = 15). the implications for identifying selection criteria for breeding to
***
Significance at the 0.1% level (DF = 15). improve NUE in future years.
 O. Gaju et al. / Field Crops Research 123 (2011) 139–152
4.1. The physiological basis of cultivar differences in NUE
4.1.1. Effects of anthesis date on grain yield and NUE
Differences in NUE were found amongst the cultivars, under
both LN and HN conditions. Since the N fertilizer amount applied
was the same for all cultivars in a given N treatment at a given site,
these differences simply reflect differences in GY in a given site/N
treatment combination. There was a positive association between
AD and GY at three sites. At NO and SB later AD was associated
with increases in both HI and AGDM, but the proportionally larger
effect was for AGDM. At CF, the overall higher GY for the cultivars
with later AD related to an effect in 2006–7 with a neutral effect
of AD on GY and NUE in 2007–8; the effect in 2006–7 was asso-
ciated with trends for increases in both HI and biomass with later
AD. In this study, cultivars with later AD tended to be feed cultivars
whereas the earlier cultivars tended to be bread-making cultivars
with lower yield potential, and this may have partly contributed
to the observed effects. Greater yield with later AD due to Ppd-
D1b was previously reported in the UK in Ppd-D1b/Ppd-D1a NILs of
Mercia associated with a decrease in HI but a proportionally greater
increase in AGDM (Foulkes et al., 2004). So the Ppd-D1b allele likely
contributed to the increase in AGDM with later AD observed in the
present study.
In the three sites that showed an association between AD and GY,
in each case the Ppd-D1b and Ppd-D1a group of cultivars responded
similarly to N supply for NUE, NUpE and NUtE. In summary, the
differential responses of cultivars to N supply observed for NUE in
the present study were not well associated with differences in AD,
e.g. contrasting NUE responses to N supply were observed for Rialto
and Robigus with the same AD; and also for Arche and Quebon with
similar AD.
4.1.2. Genetic variation in NUE in relation to NUpE and NUtE
components
Present results showed genetic variation in NUE was more
closely associated with NUtE than NUpE under both LN and HN con-
ditions. Several previous investigations worldwide indicated NUpE
accounts for a greater proportion of the genetic variation in NUE
at LN than at HN supply, e.g. amongst 10 spring wheat cultivars in
Mexico (Ortiz-Monasterio et al., 1997), 20 winter wheat cultivars
in France (LeGouis et al., 2000) and >40 spring wheat cultivars in
Finland (Muurinen et al., 2006). Present results showed there was
an association between NUpE and NUE at two sites (NO and SB)
under LN, but no association at any site under HN conditions. In
contrast, the association between NUtE and NUE was significant at
all sites under both HN and LN. Therefore, the present results show
some consistency with previous investigations in that the asso-
ciation between NUpE and NUE tended to increase as the degree
of N limitation increased (GY loss at NO was 49% compared with
9–25% at the other three sites, and Nitrogen Nutrition Index [NNI]
at anthesis under LN averaged across cultivars was 0.40 compared
to 0.45–0.54, respectively; data not shown). At NO under LN, NUpE
(R2 = 0.40) explained a broadly similar amount of the variation in
NUE as NUtE (R2 = 0.46); whereas at other sites under LN NUtE
(range R2 : 0.33–0.47) explained much more of the variation in NUE
than NUpE (range R2 : 0.01–0.17). Genetic variation in NUE associ-
ated with NUtE under LN conditions was also previously reported in
sets of historic cultivars in the UK (Foulkes et al., 1998) and France
(Brancourt-Hulmel et al., 2003). The present results are also broadly
consistent with those of Barraclough et al. (2010) who observed in
an investigation of 39 elite UK winter wheat cultivars that NUtE
was the key component determining genetic variation in GY under
either low, intermediate or high N conditions.
The N treatment × cultivar interaction for GY (or NUE) was sig-
nificant overall with Rialto (0.32) and Quebon (0.33) losing a higher
proportion of GY under LN relative to that under HN than CF9107
149
(0.25), Arche, Consort and Robigus (0.26). Cultivar differences in
ability to maintain GY under LN were partly associated with a signif-
icant N treatment × cultivar interaction for N uptake. For example,
better maintenance of GY under LN for Robigus was associated with
high relative N uptake under LN and the converse for Quebon. Over-
all therefore in the range of N limitation imposed in the present
study, GY responses of cultivars to LN did depend on responses to
both NUpE and NUtE. The mechanisms underlying the differences
amongst cultivars in NUpE cannot be certain from the present find-
ings; there were no correlations with plant height and only a weak
association for N uptake to increase with later AD as already shown
by Laperche et al. (2006). It can be speculated that increased root-
ing capacity may be associated with the cultivar differences in N
acquisition under restricted N supply, e.g. Robigus versus Quebon,
but rooting traits were not measured in the present study. Deeper
rooting might be favoured by later AD (Foulkes et al., 2004) and/or a
relatively deeper distribution of roots or a lower specific root mass
(King et al., 2003).
More generally in the LN treatment, present results showed N
recovery was probably close to 100% of that potentially available,
with NUpE ranging from 0.98 at CF to 1.76 at SB, presumably relat-
ing to differences in N mineralization of organic N during the spring
and summer after the soil mineral N was measured in February.
The highest post-February soil N mineralization appears to have
occurred at SB possibly due to the preceding crop being winter
oilseed rape in both years associated with relative high organic
residues. The average cultivar range of AGNH of 16.5 kg N ha−1
(and excluding Recital 9.9 kg N ha−1 ) was relatively small. Although
improvements in NUE seem be possible through breeding for higher
NUpE under restricted N supply as discussed above possibly involv-
ing selection for optimized rooting traits they may be relatively
modest. Turning to consider N uptake at HN, in addition to root-
ing traits, the maximum rate of N uptake (kg N m−2 d−1 ) may be a
trait that could improve uptake by allowing more N fertilizer to be
acquired shortly after N application during stem extension before
it is leached and/or immobilized in the soil organic matter (Foulkes
et al., 2009). This, in turn, may be favoured by a high storage capacity
for N in the stems (Pask, 2009).
In the present study, PANU was positively associated with GY
under LN conditions, but there was a neutral association under HN
conditions. This suggested that traits favouring PANU could be a
potential target for breeding for improved GY performance under
low to moderate N inputs. Under LN conditions, there was no asso-
ciation between PANU and AD. So apparently traits other than AD
were underlying the cultivar differences in PANU. It is possible that
variation in PANU was partly driven by above-ground N ‘demand’
according to variation in grain number per unit of ground surface
area and/or senescence-related variables (see below). Under HN
conditions, PANU showed no association with GY but was nega-
tively correlated with NUtE. PANU in these circumstances may have
represented “luxury” N uptake for the feed wheats, although it may
have played an important role in maintaining GPC in bread-making
wheats (Bogard et al., 2010). Under HN conditions, AGNA was
negatively correlated with PANU, indicating that increased N avail-
ability due to lower AGNA in the shorter phase to anthesis in early
anthesis cultivars may have partly determined the cultivar differ-
ences in PANU. N not acquired up to anthesis will be partly available
during the post-anthesis period except N that has been leached,
immobilized or, volatilized. Similar effects of phenology on AGNA
and PANU in winter wheat were previously reported (Barbottin
et al., 2005; Bogard et al., 2011).
Present results showed genetic variation in NUE was signifi-
cantly associated with NUtE at all sites under both LN and HN. These
differences, in turn, were not associated with genetic differences
in NHI but were strongly negatively associated with differences
in GPC. Numerous previous studies of cultivars and segregating
150 O. Gaju et al. / Field Crops Research 123 (2011) 139–152
populations have shown inverse relationships between GY and
GPC (e.g. Kibite and Evans, 1984; Triboï et al., 2006). The N treat-
ment × cultivar interaction for NUtE was statistically significant
with cultivars which increased NUtE relatively more under low N,
e.g., Arche and CF9107, maintaining NUE and GY relatively better
under LN. Therefore, an enhanced ability to produce viable grains
at a low GPC may be a trait associated with high GY under LN
conditions. In other words, the minimum GPC in the biomass or
grain at harvest will largely determine how much biomass or grain
can be produced per unit of absorbed N. Van Keulen (1977) con-
cluded that the minimum level to which protein can be diluted in
small grains is approximately 9.2%, and our data showed a min-
imum value of 6.4% at NO in 2006–7; a similar value of 7.2% was
reported by Martre et al. (2006). Raising NUtE associated with lower
GPC is feasible in end-use markets for which a high grain starch to
protein ratio is desirable, e.g. the feed, distilling or biofuel mar-
kets. NUtE in the present study was highly correlated with GPC, so
under moderate to low N conditions a possible constraint on rais-
ing GY may be the minimum GPC for a viable grain, and the extent
to which this may be lowered below current minimum values of
approximately 6.4%. A lower GPC could imply more photosynthe-
sis per unit of absorbed N which may, in turn, be linked to a reduced
efficiency of post-anthesis N remobilisation to the grain. The corre-
lations between senescence-related traits and crop NRE, NUtE and
GY are now discussed further below.
4.1.3. Associations between canopy senescence-related
parameters and NUE and crop NRE
The present results indicated no significant associations
between SENONSET and either GY or NUtE under HN conditions,
but associations between SENONSET and each of GY and NUtE in
three out of four sites under LN. These findings support previous
investigations showing under optimal conditions wheat crops are
in general little limited by the assimilate supply during grain fill-
ing (Dreccer et al., 1997; Borrás et al., 2004; Calderini et al., 2006).
However, under LN, present results implied GY was limited by
post-anthesis assimilate supply. Genetic variation in ‘stay-green’
(delayed senescence associated with extended photosynthesis)
was previously reported in bread wheat (Silva et al., 2000; Verma
et al., 2004; Foulkes et al., 2007). Christopher et al. (2008) found
that the stay-green phenotype in spring wheat SeriM82 was associ-
ated with extraction of deep soil water in Australia. More extensive
studies have been carried out on the mechanisms underlying
genetic variation in senescence patterns in sorghum as mentioned
above, where differences amongst sorghum hybrids were linked to
changes in the balance between N demand and supply during grain
filling.
In the present study, associations between PANU and stay-green
as indicated by SENONSET were not detected under LN. However,
there was a negative association between NRE and SENONSET , indi-
cating that post anthesis N remobilization was associated with
the observed differences in SENONSET . Previous investigations have
also reported that genetic variation in leaf senescence was asso-
ciated with post-anthesis N remobilization in maize (Masclaux
et al., 2001) and sorghum (Van Oosterom et al., 2010a,b) and that
reducing NRE might be a mechanism of delaying the onset of post-
anthesis senescence. In the present study, there were no consistent
associations between SENONSET and grain N ‘demand’ quantified
either as GN or grain number per unit AGNA ; or between SENONSET
and post-anthesis grain N source size, as indicated by AGNA .
Present results indicated that the genetic differences in
SENONSET were partly associated with differences in AD. Under
LN, there was a negative correlation between AD and SENONSET
at CF (r = −0.56, P < 0.01), but a positve correlation at NO (r = 0.66,
P < 0.001) and SB (r = 0.51, P < 0.05). The negative correlation at CF
could be attributed to two Ppd-D1a cultivars, Recital and Soisson,
each showing delayed senescence with early AD; excluding these
two Ppd-D1a cultivars (or all three Ppd-D1a cultivars) the associa-
tion between AD and SENONSET was not statistically significant. The
positive corrleations under LN at NO and SB were detected either for
all 16 cultivars or the group of 13 Ppd-D1b cutivars; and in addition
there was a positive correlation (r = 0.52, P < 0.05) amongst the 13
Ppd-D1 cutivars at EM. The reason for the prolonged canopy survival
of the two Ppd-D1a cultivars cannot be certain from the present
measurements. It was not associated with differences in PANU or
NRE; it can be speculated that maximum canopy green area was
reduced with Ppd-D1a, as reported by Foulkes et al. (2004), lead-
ing to more ‘storage N’ accumulation per unit green surface area,
thus favouring delayed senescence. Further work is needed to test
this hypothesis. With regard to the positive relationship between
AD and SENONSET within the 13 Ppd-D1b cultivars, AD was not con-
sistently associated with AGNA , PANU or the the grain N supply:
demand ratio as indicated by N accumulation at GS61 per grain.
However, there was a non-significant trend for a negative associ-
ation between AD and NRE (r = −0.36), which may have favoured
delayed senescence in late flowering cultivars. Further ecophysio-
logical studies including an analysis of N accumulation at anthesis
and N remobilization during the post-anthesis period for specific
plant components (leaf lamina, leaf sheath and stem) are needed to
further investigate the possible factors involved. In summary, our
results indicated that delayed onset of senescence favoured greater
NUE and GY under LN, but that a large proportion of the genetic vari-
ation in SENONSET appeared to be associated with AD. Nevertheless,
a stepwise multiple linear regression based on an algorithm of Afifi
and Azen (1971) applied to the data under LN indicated that a pro-
portion of the phenotypic variation in GY at LN amongst cultivars
was accounted for by SENONSET after accounting for the effect of AD;
this phenotypic variation in SENONSET independent of AD was 5.7%
at SB, 4.2% at NO and 2.2% at EM. Future work should focus on iden-
tifying sources of genetic variation in onset of senescence with a
physiological basis independent of AD, and may require character-
ization of segregating populations which are standardized for AD
as well as screening a wider range of wheat germplasm including
synthetic wheats and wheat diploid wild species for novel variation
in senescence-related traits.
4.2. Implications for breeding for NUE
Currently, in the north west Europe wheat breeding pro-
grammes cultivars are selected mostly under HN levels and it
would not be cost-effective to select NUE traits under both LN
and HN conditions at multi-location trials, although it has been
shown that direct selection at LN is more efficient (Brancourt-
Hulmel et al., 2005). In the present study, in the LN environments
NUpE was associated with phenotypic variation amongst cultivars
in NUE and GY at two sites. In order to improve NUpE, optimizing
the rooting system will be an important consideration; a recently
developed model indicated that the deeper the relative distribution
of the roots the greater the N capture as a proportion of that avail-
able to the plant (King et al., 2003). Positive associations between
increased root density at depth and N uptake in wheat has also
been demonstrated in field investigations, where variation in root-
ing traits was due to either different sowing dates and soil types
(Barraclough, 1986) or genotypes (e.g. Ford et al., 2006), and present
findings indicated it may be important to consider rooting traits
in breeding programmes for NUE. Overall, however, our results
indicated that NUtE rather than NUpE was the most important
sub-component of NUE explaining phenotypic variation amongst
genotypes in all sites. Phenotypic variation in NUtE and GY amongst
genotypes was, in turn, correlated with SENONSET under LN condi-
tions and genetic variation in SENONSET appeared to be linked to
stay-green associated with crop NRE. These findings indicate that
 O. Gaju et al. / Field Crops Research 123 (2011) 139–152
identifying the determinants of NRE and their genetic control will
be important in future strategies to increase photosynthetic capac-
ity during grain filling and NUtE in wheat breeding programmes
under low to medium N levels. Field screening for physiological
traits such as stay-green properties in wheat can be laborious and
time-consuming. Several ground-based sensors offer promise for
screening large numbers of genotypes for leaf senescence status
with high precision including those based on measuring spectral
reflectance and chlorophyll content (Babar et al., 2006). Spectral
reflectance indexes may be of particular use in obtaining an objec-
tive measure of senescence (Idso et al., 1980; Adamsen et al., 1999;
Gupta et al., 2001). In current experiments, the correlation amongst
genotypes between SENONSET measured under LN and HN was not
statistically significant in three out of the four sites, indicating a
significant N treatment × genotype interaction. Thus, to be repre-
sentative of low N performance, assessment of this trait must be
carried out at low N supply. Further studies are required combining
genetics and physiology to identify the location of QTLs for senes-
cence related traits linked to NUtE and to better understand the
way in which alleles associated with QTL may promote substantial
differences in NUE in winter wheat.
Acknowledgements
The authors thank BBSRC and INRA for funding. We thank
Damien Bouthors, Dominique Brasseur, Benoît Dugué, Jean-Pierre
Noclercq (INRA Etrées-Mons), Jean-Louis Joseph, Pascal Lemaire,
Joëlle Messaoud, Nicole Allard and Bernard Bonnemoy (INRA
Clermont-Ferrand), John Alcock and Matthew Tovey (University of
Nottingham) for their assistance with experiments.
References
Adamsen, F.J., Pinter, P.J., Barnes, E.M., LaMorte, R.L., Wall, G.W., Leavitt, S.W., Kim-
ball, B.A., 1999. Measuring wheat senescence using a digital camera. Crop Sci.
39, 719–724.
Afifi, A.A., Azen, S.P., 1971. Statistical Analysis, A Computer Aided Approach. Aca-
demic, London.
Anbessa, Y., Juskiw, P., Good, A., Nyachiro, J., Helm, J., 2009. Genetic variability in
nitrogen use efficiency of spring barley. Crop Sci. 49, 1259–1269.
Babar, M.A., Reynolds, M.P., Van Ginkel, M., Klatt, A.R., Raun, W.R., Stone, M.L., 2006.
Spectral reflectance to estimate genetic variation for in-season biomass, leaf
chlorophyll, and canopy temperature in wheat. Crop Sci. 46, 1046–1057.
Barbottin, A., Lecomte, C., Bouchard, C., Jeuffroy, M.H., 2005. Nitrogen remobilization
during grain filling in wheat: genotypic and environmental effects. Crop Sci. 45,
1141–1150.
Barraclough, P.B., 1986. The growth and activity of winter wheat roots in the field:
nutrient uptakes of high-yielding crops. J. Agric. Sci. (Camb.) 106, 45–52.
Barraclough, P.B., Howarth, J.R., Jones, J., Lopez-Bellido, R., Parmar, S., Shepherd, C.E.,
Hawkesford, M.J., 2010. Nitrogen efficiency of wheat: genotypic and environ-
mental variation and prospects for improvement. Eur. J. Agron. 33, 1–11.
Benesch, R., Mangelsdorf, P., 1972. A method for the colorimetric determination of
ammonia in seawater. Helgoländer Wiss Meeresunters 23, 365–375.
Berry, P.M., Spink, J., Foulkes, M.J., White, P.J., 2010. The physiological basis of geno-
typic differences in nitrogen use efficiency in oilseed rape (Brassica napus L.).
Field Crops Res. 119, 365–373.
Bertin, P., Gallais, A., 2000. Physiological and genetic basis of nitrogen use efficiency
in maize. I. Agro-physiological results. Maydica 45, 53–60.
Bogard, M., Allard, V., Brancourt-Hulmel, M., Heumez, E., Machet, J.-M., Jeuffroy,
M.-H., Gate, P., Martre, P., LeGouis, J., 2010. Deviations from the grain protein
concentration – grain yield negative relationship are highly correlated to post-
anthesis N uptake in winter wheat. J. Exp. Bot., 10.1093/jxb/erq1238.
Bogard, M., Jourdan, M., Allard, V., Martre, P., Perretant, M.-R., Ravel, C., Heumez, E.,
Orford, S., Snape, J., Griffiths, S., Gaju, O., Foulkes, J., Le Gouis, J., 2011. Anthesis
date mainly explained correlations between post-anthesis leaf senescence, grain
yield, and grain protein concentration in a winter wheat population segregating
for flowering time QTLs. J. Exp. Bot., doi:10.1093/jxb/err061.
Borras, L., Slafer, G.A., Otegui, M.E., 2004. Seed dry weight response to source–sink
manipulations in wheat, maize and soybean: a quantitative reappraisal. Field
Crop Res. 86, 131–146.
Borrell, A.K., Hammer, G.L., 2000. Nitrogen dynamics and the physiological basis of
stay-green in sorghum. Crop Sci. 40, 1295–1307.
Brancourt-Hulmel, M., Doussinault, G., Lecomte, C., Berard, P., Le Buanec, B., Trottet,
M., 2003. Genetic improvement of agronomic traits of winter wheat cultivars
released in France from 1946 to 1992. Crop Sci. 43, 37–45.
151
Brancourt-Hulmel, M., Heumez, E., Pluchard, P., Béghin, D., Depatureaux, C., Giraud,
A., LeGouis, J., 2005. Indirect versus direct selection of winter wheat for low
input or high input levels. Crop Sci. 45, 1427–1431.
Calderini, D.F., Reynolds, M.P., Slafer, G.A., 2006. Source–sink effects on grain weight
of bread wheat, durum wheat, and triticale at different locations. Aust. J. Agric.
Res. 57, 227–233.
Christopher, J.T., Manschadi, A.M., Hammer, G.L., Borell, A.K., 2008. Developmen-
tal and physiological traits associated high yield and stay-green phenotype in
wheat. Aust. J. Agric. Res. 59, 354–364.
Dhugga, K.S., Waines, J.G., 1989. Analysis of nitrogen accumulation and use in bread
and durum wheat. Crop Sci. 29, 1232–1239.
Dreccer, M.F., 2005. Nitrogen use at the leaf and canopy level: a framework to
improve NUE. J. Crop Impr. 15, 97–128.
Dreccer, M.F., Grashoff, C., Rabbinge, R., 1997. Source-sink ratio in barley (Hordeum
vulgare L.) during grain filling: effects on senescence and grain protein concen-
tration. Field Crop Res. 49, 269–277.
Ford, K.E., Gregory, P.J., Gooding, M.J., Pepler, S., 2006. Genotype and fungicide effects
on late-season root growth of winter wheat. Plant Soil 284, 33–44.
Foulkes, M.J., Sylvester-Bradley, R., Scott, R.K., 1998. Evidence for differences
between winter wheat cultivars in acquisition of soil mineral nitrogen and
uptake and utilization of applied fertiliser nitrogen. J. Agric. Sci. (Camb.) 130,
29–44.
Foulkes, M.J., Sylvester-Bradley, R., Worland, A., Snape, J., 2004. Effects of a photope-
riod sensitivity gene Ppd-D1 on yield potential and drought resistance in UK
winter wheat. Euphytica 135, 63–74.
Foulkes, J., Holdsworth, M., Kerr, S., Kightly, S., Barraclough, P., Hawkesford, M.,
Shewry, S., 2006. A study of the scope for the application of crop genomics and
breeding to increase nitrogen economy within cereal and rapeseed based food
chains. Final Report for Project AR0714. Defra, London.
Foulkes, M.J., Sylvester-Bradley, R., Weightman, R., Snape, J.W., 2007. Identifying
physiological traits associated with improved drought resistance in winter
wheat. Field Crop Res. 103, 11–24.
Foulkes, M.J., Reynolds, M.P., Sylvester-Bradley, R., 2009. Genetic improvement of
grain crops: yield potential. In: Sadras, V.O., Calderini, D.F. (Eds.), Crop Physi-
ology Applications for Genetic Improvement and Agronomy. Academic Press,
Amsterdam, pp. 355–386.
Génard, M., Reich, M., Lobit, P., Besset, J., 1999. Correlations between sugar and acid
content and peach growth. J. Hortic. Sci. Biotechnol. 74, 772–776.
Gupta, R.K., Vijayand, D., Prasad, T.S., 2001. Characterisation of red-near infrared
transition for wheat and chickpea using 3 nm bandwidth data. Adv. Space Res.
28, 89–194.
Hirel, B., LeGouis, J., Ney, B., Gallais, A., 2007. The challenge of improving nitrogen
use efficiency in crop plants: towards a more central role for genetic variability
and quantitative genetics within integrated approaches. J. Exp. Bot. 58, 2369–
2387.
Horst, W.J., Behrens, T., Heuberger, H., Kamh, M., Reidenbach, G., Wiesler, F., 2003.
Genotypic differences in nitrogen use-efficiency in crop plants. In: Lynch, J.M.,
Schepers, J.S., Ünver, I. (Eds.), Innovative Soil-plant Systems for Sustainable Agri-
cultural Production. , pp. 75–92.
Idso, S.B., Pinter, P.J., Jackson, R.D., Reginato, R.J., 1980. Estimation of grain yields by
remote sensing of crop senescence rates. Remote Sens. Environ. 9, 87–91.
Justes, E., Mary, B., Meynard, J.M., Machet, J.M., Thelier-Huche, L., 1994. Determina-
tion of a critical nitrogen dilution curve for winter wheat crops. Ann. Bot. 74,
397–407.
Kibite, S., Evans, L.E., 1984. Causes of negative correlations between grain yield and
grain protein concentration in common wheat. Euphytica 33, 801–810.
King, J., Gay, A., Sylvester-Bradley, R., Bingham, I., Foulkes, J., Gregory, P., Robinson,
D., 2003. Modelling cereal root systems for water and nitrogen capture towards
an economic optimum. Ann. Bot. 91, 383–390.
Laperche, A., Brancourt-Hulmel, M., Heumez, E., Gardet, O., LeGouis, J., 2006. Esti-
mation of genetic parameters of a DH wheat population grown at different N
stress levels characterized by probe genotypes. Theor. Appl. Genet. 112, 797–
807.
LeGouis, J., Beghin, D., Heumez, E., Pluchard, P., 2000. Genetic differences for nitrogen
uptake and nitrogen utilisation efficiencies in winter wheat. Eur. J. Agron. 12,
163–173.
Martre, P., Jamieson, P.D., Semenov, M.A., Zyskowski, R.F., Porter, J.R., Triboi, E.,
2006. Modelling protein content and composition in relation to crop nitrogen
dynamics for wheat. Eur. J. Agron. 25, 138–154.
Masclaux, C., Quillere, I., Gallais, A., Hirel, B., 2001. The challenge of remobilisa-
tion in plant nitrogen economy. A survey of physio-agronomic and molecular
approaches. Ann. Appl. Biol. 138, 69–81.
Mi, C., Liu, J., Chen, F., Zhang, F., Cui, L., Liu, X., 2003. Nitrogen uptake and remobi-
lization in maize hybrids differing in leaf senescence. J. Plant Nutr. 26, 447–459.
Moll, R.H., Kamprath, E.J., Jackson, W.A., 1982. Analysis and interpretation of factors
which contribute to efficiency to nitrogen utilization. Agron. J. 75, 562–564.
Mortimer ND, Elsayed MA, Horne RE, 2004. Energy and greenhouse gas emissions
for bioethanol production from wheat grain and sugar beet. York, UK: NNFC:
Report for British Sugar no. 23/1.
Muurinen, S., Slafer, G.A., Peltonen-Sainio, P., 2006. Breeding effects on nitrogen use
efficiency of spring cereals under northern conditions. Crop Sci. 46, 561–568.
Ortiz-Monasterio, J.I., Sayre, K.D., Rajaram, S., McMahom, M., 1997. Genetic progress
in wheat yield and nitrogen use efficiency under four nitrogen rates. Crop Sci.
37, 898–904.
Oury, F.-X., Bérard, P., Brancourt-Hulmel, M., Depatureaux, C., Doussinault, G., Galic,
N., Giraud, A., Heumez, E., Lecomte, C., Pluchard, P., Rolland, B., Rousset, M.,
152 O. Gaju et al. / Field Crops Research 123 (2011) 139–152
Trottet, M., 2003. Yield and grain protein concentration in bread wheat: a review
and a study of multi-annual data from a French breeding program. J. Genet.
Breeding 57, 59–68.
Pask, A.J.D., 2009. Optimising nitrogen storage in wheat canopies for genetic reduc-
tion in fertiliser nitrogen inputs. Ph.D. Thesis. University of Nottingham, UK.
Rémy, J.C., Hébert, J., 1977. Le devenir des engrais dans le sol. Compte Rendu de
l’Académie d’Agriculture de France 63, 700–710.
Silva, S.A., de Carvalho, F.I.F., Caetano, V.R., Oliveira, A.C., Coimbra, J.L.M., Vasconcel-
los, N.J.S., Lorencetti, C., 2000. Genetic basis of stay-green trait in bread wheat.
J. New Seeds 2, 55–68.
Sylvester-Bradley, R., Kindred, D.R., 2009. Analysing nitrogen responses of cereals to
prioritize routes to the improvement of nitrogen use efficiency. J. Exp. Bot. 60,
1939–1951.
Triboï, E., Martre, P., Girousse, C., Ravel, C., Triboï-Blondel, A.M., 2006. Unravel-
ling environmental and genetic relationships between grain yield and nitrogen
concentration for wheat. Eur. J. Agron. 25, 108–118.
Thomas, H., Smart, C.M., 1993. Crops that stay green. Ann. Appl. Biol. 123, 193–219.
Van Oosterom, E.J., Borrell, A.K., Chapman, S.C., Broad, I.J., Hammer, G.L., 2010a. Func-
tional dynamics of the nitrogen balance of sorghum: I. N demand of vegetative
plant parts. Field Crop Res. 115, 19–28.
Van Oosterom, E.J., Chapman, S.C., Borrell, A.K., Broad, I.J., Hammer, G.L., 2010b.
Functional dynamics of the nitrogen balance of sorghum: II. Grain filling period.
Field Crop Res. 115, 29–38.
Van Keulen, H., 1977. Nitrogen Requirements of Rice with Special Reference to Java
Bogor. Ministry of Agriculture, Agricultural Technical Assistance Programme,
Indonesia, 67 pp.
Verma, V., Foulkes, M.J., Caligari, P., Sylvester-Bradley, R., Snape, J., 2004. Mapping
quantitative trait loci for flag leaf senescence as a yield determinant in win-
ter wheat under optimal and drought-stressed environments. Euphytica 135,
255–263.
Wagner, R., 1974. A new method for automated nitrate determination in sea water
using the autoAnalyzer. In: Technicon Symposium , Frankfurt a. M.
Zadoks, J.C., Chang, T.T., Konzak, C.F., 1974. A decimal code for the growth stages of
cereals. Weed Res. 14, 415–421.