Research
Modelling postsilking nitrogen fluxes in maize (Zea mays)
using 15N-labelling field experiments
Blackwell Publishing Ltd
André Gallais1,2, Marie Coque1, Isabelle Quilléré3, Jean-Louis Prioul4 and Bertrand Hirel3
1
Station de Génétique Végétale, INRA-UPS-INAPG-CNRS, Ferme du Moulon, 91190 Gif/Yvette, France; 2INAPG, 16 rue Claude Bernard, 75231 Paris
Cedex 05, France; 3Unité de Nutrition Azotée des Plantes, INRA route de St Cyr, 78026 Versailles Cedex, France; 4Institut de Biotechnologie des Plantes,
Université de Paris-Sud, 91405 Orsay Cedex, France
Summary
Author for correspondence:
André Gallais
Tel: +33 169332331
Fax: +33 169332340
Email: [email protected]
Received: 30 June 2006
Accepted: 7 August 2006
• In maize (Zea mays), nitrogen (N) remobilization and postflowering N uptake are
two processes that provide amino acids for grain protein synthesis.
• To study the way in which N is allocated to the grain and to the stover, two
different 15N-labelling techniques were developed. 15NO3− was provided to the soil
either at the beginning of stem elongation or after silking. The distribution of 15N in
the stover and in the grain was monitored by calculating relative 15N-specific
allocation (RSA).
• A nearly linear relationship between the RSA of the kernels and the RSA of the stover
was found as a result of two simultaneous N fluxes: N remobilization from the stover to
the grain, and N allocation to the stover and to the grain originating from N uptake.
• By modelling the 15N fluxes, it was possible to demonstrate that, as a consequence
of protein turnover, a large proportion of the amino acids synthesized from the N
taken up after silking were integrated into the proteins of the stover, and these
proteins were further hydrolysed to provide N to the grain.
Key words: maize (Zea mays), modelling, 15N labelling, nitrogen remobilization,
nitrogen uptake, nitrogen use efficiency, protein turnover.
New Phytologist (2006) 172: 696–707
© The Authors (2006). Journal compilation © New Phytologist (2006)
doi: 10.1111/j.1469-8137.2006.01890.x
Introduction
Nitrogen (N) is one of the main limiting factors for plant
growth and ultimately for the production of harvestable
plant material used for animal and human food. In most
plant species examined so far, the plant life cycle with regard to
the management of N can be roughly divided into two main
phases occurring successively. During the first phase, i.e. the
vegetative phase, young developing roots and leaves behave as
sink organs for the assimilation of inorganic N and the
synthesis of amino acids. These amino acids are further used
for the synthesis of enzymes and proteins mainly involved in
building up plant architecture and the different components
of the photosynthetic machinery. Notably, the enzyme
Rubisco can alone account for up to 50% of the total soluble
leaf protein content (Mae et al., 1983). Later, at a certain stage
696
of plant development generally starting after anthesis, the
remobilization of N takes place. At this stage, shoots and/or
roots start to behave as sources of N by providing amino acids
released from protein hydrolysis, which are subsequently exported
to reproductive and storage organs represented, for example,
by seeds, bulbs or trunks (Masclaux et al., 2001). In maize,
45–65% of the grain N is provided from pre-existing N in the
stover before silking, a process that is strongly dependent upon
the environmental conditions and/or the genotype (Weiland
& Ta, 1992; Gallais & Coque, 2005). The remaining 35–55%
of the grain N originates from postsilking N uptake (Ta &
Weiland, 1992; Bertin & Gallais, 2000; Gallais & Coque, 2005).
Therefore, two distinct N fluxes must be considered during
the grain-filling period: N translocation from the stover
(stalks + leaves + sheaths + cob) and N uptake (Crawford et al.,
1982; Rendig & Crawford, 1985; Ta & Weiland, 1992).
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However, from the point of view of N management at the
whole-plant or organ level, the arbitrary separation of the
plant life cycle into two phases is rather simplistic, as it is
well known that, for example, N recycling can occur before
anthesis for the synthesis of new proteins in developing organs
(Lattanzi et al., 2005). In addition, during the assimilatory
phase, the ammonium incorporated into free amino acids is
subjected to a high turnover, as a result of photorespiratory
activity, as it needs to be immediately re-assimilated into
glutamine and glutamate (Hirel & Lea, 2001; Novitskaya et al.,
2002). Therefore, the photorespiratory flux of ammonium,
which, at least in C3 plants, can be 10 times higher than that
originating from nitrate reduction, is mixed with that channelled
through the inorganic N assimilatory pathway (Novitskaya
et al., 2002). Although in C4 plants photorespiration is low,
this process is likely to occur in maize (Zea mays) (Taiz &
Zeiger, 2002; Ueno et al., 2005). Furthermore, Liu & Jagendorf
(1984) and Malek et al. (1984) found that in pea (Pisum sativum)
approx. 30% of the labelled amino acids newly incorporated
into proteins by isolated chloroplasts were lost during the next
30 min, after a chase with cold amino acids. Therefore, the
occurrence of protein turnover concomitantly with the two
fluxes of ammonium (from assimilatory and photorespiratory
fluxes) introduces another level of complexity in the exchange
of N within the pool of free amino acids. The coexistence of
these different N fluxes has led us to reconsider the mode
by which N is managed from the cellular level to the level of
the whole plant (Hirel & Gallais, 2006).
Despite the complexity of the N fluxes, a number of
attempts have been made to estimate globally the amount of
N remobilized to the grain. To achieve this, methods based on
the calculation of N budgets, termed the ‘balance method’, or
techniques using 15N-labelling have been used. The ‘balance
method’ allows estimation of the amount of N remobilized by
calculating the difference between total plant N (stalks +
leaves) at silking and total N in the stover at grain maturity.
This method is based on the assumption that all the newly
synthesized amino acids originating from N uptake are
directly allocated to the grain (Moll et al., 1982; Di Fonzo
et al., 1993; Rajcan & Tollenaar, 1999; Bertin & Gallais,
2000). However, this method does not take into account the
possibility that a significant amount of the newly synthesized
amino acids may be allocated to the stover before leaf N
remobilization, as already shown by Ta & Weiland (1992).
The other techniques, which use 15N-labelled fertilizer, provide an elegant way of investigating how N originating from
postsilking uptake is directed towards the kernels in the grain.
Methods based on short- or long-term labelling have been
developed using plants grown either under hydroponics or
in the field. Hydroponics allows 15N application during a
well-defined period, either short (with pulse chase experiments)
or long (with quasi steady-state labelling), whereas in the field
only long-term labelling is possible, with the problem that
some residual 15N can remain in the soil long after the
labelling period. However, the benefit of field experiments
employing long-term labelling near natural abundance is
that they allow the separation of newly assimilated N from
that taken up earlier, as discussed by Deléens et al. (1994).
Furthermore, a large number of genotypes can be studied
under agronomic conditions.
N recovery from fertilizer has been studied in 15N-labelling
experiments in the field (for example, in wheat (Triticum
aestivum) by Broadbent & Carlton, 1978, and in maize by
Ma & Dwyer, 1998). The reallocation of N to the different
organs has been analysed following application of the isotope
at different plant developmental stages in maize in the field
(Ta & Weiland, 1992; Ma & Dwyer, 1998) or in hydroponics
(Weiland, 1989; Cliquet et al., 1990a; Deléens et al., 1994). More
recently, Sheehy et al. (2004a,b) have used 15N-labelling in
irrigated plots to determine the temporal origin of N in the
rice (Oryza sativa) grain.
In maize, application of 15N-nitrate at the beginning of
stem elongation allows the determination of N remobilization
(Cliquet et al., 1990b). Indeed, when 15N tracer is applied
during the vegetative growth period, just before the beginning
of rapid growth resulting from stem elongation, both leaves
and stalks are labelled, whereas kernels are labelled only as a
result of N remobilization. Furthermore, if the proportion of 15N
uptake after silking is negligible (which is easier to arrange in
a hydroponic experiment than in the field), N remobilization
from the stover to the kernels can then be estimated from the
percentage of whole-plant 15N uptake allocated to the kernels.
Conversely, when the 15N tracer is applied just after silking,
the allocation of recent N uptake to the stover and to the
kernels can be estimated. Therefore, the use of two labelling
techniques at different times is desirable, in order to obtain a
complete picture of N management and recycling during the
entire developmental cycle of the maize plant.
With plants grown in the field, the originality of our study
was to use two 15N-labelling techniques (labelling at the
beginning of stem elongation and after silking), in order to
investigate the different N fluxes occurring within the plant.
A detailed analysis and exploitation of the 15N-labelling data,
taking into account the potential of the technique, were
performed by estimating the relative 15N-specific allocation
(RSA), in order to follow the fate of N assimilated after silking
and the N remobilization flux. A predictive model depicting
the 15N fluxes occurring between the stover and the grain was
then developed to explain and predict the contribution of the
various N fluxes to the amount of N in the stover protein and
to grain N protein deposition.
Materials and Methods
Field experiments
Three separate 15N-labelling experiments were performed in
the field over a 3-year period from 2001 to 2003. Compared
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New Phytologist (2006) 172: 696– 707
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with experiments conducted under controlled conditions
(Cliquet et al., 1990a,b), the main difficulty of carrying out
15
N labelling during vegetative growth was to avoid 15N
uptake after silking. To minimize the uptake of 15N label after
silking and to simultaneously favour its distribution within
the vegetative organs (leaves and stalks), a temporally discrete
application of 15N-labelled fertilizer was made at the beginning
of the rapid growth associated with stem elongation (V6
stage). Under our experimental conditions, there was an
interval of 30–37 d between the 15N application and the
silking period.
Experiment performed in 2001 The experiment was performed
to study N partitioning and management in the maize (Zea
mays L.) single cross hybrid (Déa) in comparison to its parental
lines F2 and Io. These three genotypes are commonly used at
Institut National de la Recherche Agronomique for both
physiological and molecular genetic studies (Hirel et al., 2005).
Each genotype was grown in one plot of approx. 550 m2, at
a density of 120 000 plants ha−1, in 80-cm-spaced rows and
at two levels of N fertilization: a low level (N0, 30 kg N ha−1)
and a high level (N1, 170 kg N ha−1). For each treatment
(genotype × level of N fertilization), the 15N-labelled fertilizer
was provided at the beginning of stem elongation on three
individual microplots containing 12 consecutive plants in a row.
Using a syringe, 1.25 mg of 15N was provided to each individual
plant by applying 200 ml of a solution of KNO3 at 1.94%
15
N atom excess. Labelled plants were harvested at three different
developmental stages: silking, 35 days after silking (DAS) and
grain maturity, corresponding to 60–70 DAS. For each stage
of plant development, six plants exhibiting a homogeneous
pattern of development were selected from the 12 labelled
plants in a microplot. Two plants were pooled, to obtain three
replicates. For each of the three replicates, leaves and stalks +
sheaths at silking and leaves, stalks + sheaths, husks + cobs and
kernels at maturity were separated for the analysis of dry
matter content, total N content, and 15N abundance.
Experiment performed in 2002 In this experiment, N
partitioning and management were studied using four
commercial hybrids: Déa, Anjou 285, Nicco and Tarro
(Pommel et al., 2005). 15N-labelled fertilizer was provided at
the beginning of stem elongation and at silking. The protocol
was similar to that used in 2001, except that the plant density
was 100 000 plants ha−1 and only the N1 fertilization regime
was used, with four replicates instead of three. For the
labelling during stem elongation, in each replicate the
15N-labelled fertilizer was distributed on three noncontiguous
microplots containing four plants in a row, each microplot
corresponding to a stage of harvest: silking, 35 DAS or
maturity. For the labelling at silking, four microplots were
used instead of three, as the plants were harvested 15, 25 and
35 DAS and at maturity. Each individual plant was provided
with 2.5 mg of 15N by applying 200 ml of a solution of
New Phytologist (2006) 172: 696–707
KNO3 at 4.91% 15N atom excess. In the two labelling
experiments and for each stage of plant development, two
labelled plants exhibiting the same phenotype were selected
from each microplot. Leaves and stalks + sheaths at silking
and leaves, stalks + sheaths, husks + cobs and kernels at
maturity were separated for the analysis of dry matter content,
total N content, and 15N abundance.
Experiment performed in 2003 The aim of this third
experiment was to verify the results obtained in the postsilking 15N-labelling experiment performed in 2002. Two
experimental hybrids randomly chosen among the 100
examined by Bertin & Gallais (2000) and Coque (2006) were
studied at four stages of plant development including silking,
15 DAS, 25 DAS and maturity. The experimental protocol
was similar to that used in 2002 except that the 2.5 mg 15N
applied per plant was dissolved in 3 l and three applications of
1 l each were performed every 3 d over a 9-d period to favour
distribution within the soil. At silking, 15 DAS and 25 DAS,
for each of the three replicates, 15N was provided to
microplots of six consecutive plants in a row 5 m long. Three
plants exhibiting similar phenotypes were harvested for 15N
analysis. For the sampling at maturity, for each of the two
replicates 15N was provided to microplots of eight plants, with
six plants being harvested (Coque, 2006).
Determination of 15N abundance
In all experiments, after drying and weighing of each plant
part, the material was ground to obtain a homogeneous fine
powder. A subsample of 2.5 mg was used to determine total
N content and 15N abundance using an elemental analyser
(N-analyser NA1500; Carlo-Erba, Milan, Italy) coupled to an isotope ratio mass spectrometer (Optima; Micromass, Manchester,
UK) calibrated for measuring 15N natural abundance. Using
the formula δ15N = (15N/14N − 1) × 1000, the 15N abundance
(A) was calculated to determine the 15N content of the
sample using the following equation: [15N/(15N + 14N)].
For each plant organ, termed X, the atom percentage excess
(EX = AX% − A0X%) was then calculated, AX% representing
the 15N abundance percentage in the organ X considered
and A0X% representing the natural 15N abundance percentage
of the same organ X. A0X% was close to 0.36634, a value
which corresponds to the natural abundance of atmospheric
dinitrogen (N2). From E X, the relative specific allocation
(RSAX) was derived according to the method developed by
Cliquet et al. (1990a,b) and Deléens et al. (1994):
RSA X =
AX % − A0 X %
,
AS % − A0 X %
or as a percentage RSAX(%) = 100 RSAX. In our study the
organ X corresponds to the grain, the stover or the whole plant
and AS represents the 15N abundance of the labelled fertilizer.
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For a given organ, the RSA can be roughly defined as the ratio
of the 15N content originating from the labelled fertilizer to
the total N content. In order to verify whether or not 15N was
taken up after silking, when the labelling was performed at
the beginning of stem elongation, the amount of 15N (qX)
originating from the labelled fertilizer was calculated at
flowering and at maturity using the formula:
qX = Q XRSAX
(Q X, the total amount of N present in the organ X).
Statistical analyses
In each of the three experiments, to test whether genotypic
effects were statistically significant with respect to the values
obtained for both the RSAs and for the ratios RSAgrain/
RSAstover and RSAgrain/RSAwhole-plant, analyses of variance were
carried out. Either the residual error of such analyses or the
coefficient of variation was used to obtain a value for the
degree of accuracy associated with individual RSA values or
with ratios of RSAs. Furthermore, to study the relationship
between RSAgrain and RSAstover or RSAwhole-plant using all
samples, we calculated the linear regression of RSAgrain on
RSAwhole-plant (with or without the constraint of intercept
equal to zero) and the correlation r between these two values.
The significance of the correlation coefficient is indicated in
the text by *, ** and *** at a threshold of 0.05, 0.01 and 0.001,
respectively.
Modelling 15N fluxes
For modelling the 15N fluxes using the results obtained from
the different labelling experiments performed before and after
silking, it was assumed that: (1) there was no discrimination
between 15N and 14N for N distribution within the plant, or
if there was some discrimination, the impact was negligible;
(2) for labelling at the beginning of the stem elongation,
the 15N was homogeneously distributed within the plant,
proportionally to the N content of each organ, and isotopic
equilibrium was reached (see Appendix); (3) for the labelling
just after silking, the 15N was taken up proportionally to the
N originating from the soil and from the fertilizer.
The theoretical basis of the model is shown in Fig. 1, when
two main plant compartments (stover and grain) are considered. For a given genotype it was assumed (1) that the
whole-plant N content at silking (Q0) is known and (2) that
at any time t, the amount of unlabelled N taken up after
silking (At) and the amount of grain N (Gt) are also known.
The parameter x corresponds to the proportion of the newly
synthesized amino acids and proteins originating from postsilking N uptake which is allocated to the grain. Thus 1 − x
corresponds to the proportion of newly synthesized Ncontaining molecules (mainly amino acids) allocated to the
Fig. 1 The two-compartment model to explain the postsilking
nitrogen (N) fluxes in grain of maize (Zea mays). A represents the
nitrogen uptake, Q is the N quantity in the stover, G is the amount of
N in the grain, R is the N transferred from the stover to the grain, Q0
(q0) is the amount of N (quantity of 15N) at silking, q (g) is the quantity
of 15N in the stover (in the grain), and x is the proportion of amino
acids synthesized from newly taken up N and allocated to the grain.
With 15N labelling at stem elongation, q0 is different from 0, whereas
it is 0 for 15N labelling just after silking. Broken arrows represent
protein turnover.
stover, which are mixed with pre-existing proteins before N
remobilization (Rt) as a consequence of protein turnover.
To simplify the calculation, we have considered that x
remains constant whatever the plant developmental stage.
The following equation is then obtained:
R t = Gt − xA t
The quantity of N present in the stover (Qt) can be calculated
as follows:
Q t = (1 − x)At + Q0 − R t
Let gt and qt be the amount of N arising from the labelled
fertilizer and allocated to the grain and to the stover,
respectively, at time t. For modelling variation of these amounts
during grain filling, it is necessary to distinguish between the
labelling during vegetative growth and the labelling at silking.
For labelling at the beginning of stem elongation, and
assuming that there is no 15N uptake after silking, the increase
in the amount of grain 15N during a short interval of time dt
originating from the fertilizer must be equal to the decrease
in the stover. Thus, we arrive at the following differential
equation:
dg
dq dR q t
.
=−
=
dt
dt
dt Q t
In this equation, qt /Qt represents the relative amount of N
originating from the 15N-labelled fertilizer which is allocated
to the grain via N-remobilization from the stover (R). This
corresponds to the RSA of the stover at time t. Therefore,
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Table 1 Observed values for maize (Zea mays) whole-plant nitrogen
(N) uptake and N accumulated in the kernels according to stage of
plant developmental and year, utilised in the modelling of 15N fluxes
2001
2002
Stage
Whole plant
Kernels
Whole plant
Kernels
Silking
15 DAS
25 DAS
35 DAS
Maturity
1.15 ± 0.04a
–
–
1.54 ± 0.06
1.62 ± 0.11
0
–
–
0.85 ± 0.03
1.10 ± 0.05
1.49 ± 0.07
1.81 ± 0.12
1.87 ± 0.08
1.97 ± 0.12
2.28 ± 0.09
0
0.22 ± 0.03
0.35 ± 0.04
0.89 ± 0.06
1.50 ± 0.07
Data are expressed in grams of nitrogen (N) per plant, for the hybrid
Déa grown under a high N fertilization regime.
DAS, days after silking; a, standard deviation.
when Rt, Qt and q0 are known, it becomes possible to derive
gt and qt by numerical integration. A 1-d interval was used for
the integration. The amount of 15N present in the whole plant
(qwp) can then be determined as the sum of qt + gt. At any
date, the (g/Qg) and (qwp/Qwp) ratios correspond to the RSA
for the grain and to the RSA for the whole plant, respectively,
and therefore the ratio RSAgrain:RSAwhole-plant can be calculated.
For 15N labelling at silking it is necessary to define the
dynamics of 15N uptake (at). The simplest model is to assume
that 15N originating from labelled fertilizer is taken up
proportionally to the 14N present in the soil. Thus at = k A t.
This proportionality is based on the assumption that (1) 15N
is homogeneously distributed in the soil compartment
prospected by the roots and (2) there is no isotopic discrimination. Then, the differential equations become:
dg
da dR q t
=x
+
dt
dt
dt Q t
dq
da dR q t
= (1 − x ) −
.
dt
dt
dt Q t
Thus, if A t, R t and Qt are known it becomes possible to derive
gt and qt by numerical integration by considering that
q0 = 0. As described above, RSAgrain, RSAwhole-plant and the
RSAgrain:RSAwhole-plant ratio can be derived at any time t.
For the application of the model, from our 15N-labelling
experiments, we have used curves for A t (postsilking N
uptake) and Gt (N accumulated into the kernels) in such a
way that they fit the 15N-labelling experimental data obtained
with the hybrid Déa in 2001 and 2002 (Table 1). In 2001,
three harvesting periods were available: silking, 35 DAS and
maturity. Therefore, we have linearly interpolated between
silking and 35 DAS, and curvilinearly interpolated between
35 DAS and maturity. In 2002, we have only linearly
interpolated between each of the five labelling periods. The
application of the model was first developed using the Microsoft Excel spreadsheet (Microsoft 98). By assigning different
values to parameter x between 0 and 1, it was possible to
identify the value that best simulated the observed values for
the RSAgrain:RSAwhole-plant ratio. We have considered such a
ratio because of its properties, which are described in the
Results section (low coefficient of variation and value close to
1 at maturity).
Results
Variation in RSA values and relationship between
RSAgrain and RSAstover or RSAwhole-plant
Labelling during vegetative growth At silking in the 2001
experiment, for the whole-plant RSA the effect of the level of
N fertilization (N) was highly significant. The genotype × N
interaction was also significant, whereas the genotypic effect
was not significant (Table 2). The genotype × N interaction
was attributable to the genotype Io under high N fertilization,
which had a lower value than expected based on the addition
of the effects of the genotype and of the level of N fertilization. In the 2002 experiment, the genotype effect was not
significant (Table 3).
At maturity in the 2001 and 2002 experiments, we
observed a large environmental variation in RSA between
replicates, illustrated by an important residual variation in the
Table 2 Analysis of variance of relative 15N-specific allocation (RSA) in maize (Zea mays) for the whole plant and plant parts, and RSA ratios,
at silking and maturity from the results obtained in 2001 following 15N labelling at the beginning of stem elongation
Effect
RSAwhole-plant
at silking
RSAgrain
at maturity
RSAstover
at maturity
RSAwhole-plant
at maturity
RSAg:RSAsta
RSAg:RSAwpa
Genotype (G)
Nitrogen (N)
G×N
Coefficient of variation (%)
ns
***
**
16.9
ns
ns
ns
30.9
ns
ns
ns
31.3
ns
ns
ns
30.9
*
**
*
4.8
***
***
(*)
1.9
Significance: (*), P < 0.10; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.
a
RSAg, RSAgrain; RSAst, RSAstover; RSAwp, RSAwhole-plant.
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Table 3 Relative 15N-specific allocation (RSA) values at silking and maturity for the 2002 experiment following 15N labelling at the beginning
of stem elongation
Genotype
RSA at
silking (%)
RSAstover at
maturity (%)
RSAgrain at
maturity (%)
RSAwhole-plant
at maturity (%)
RSAgrain:RSAwhole-plant
at maturity
Anjou
Déa
Nicco
Tarro
Mean
F-test
Coefficient of variation (%)
1.03
1.02
0.85
0.95
0.96
ns
17.3
0.97
0.85
0.93
0.88
0.91
ns
23.0
0.96
0.85
1.03
0.93
0.94
ns
25.4
0.96
0.85
0.99
0.91
0.93
ns
24.4
1.00
1.00
1.03
1.02
1.01
(*)
1.8
Significance: (*), P < 0.10; ns, not significant.
Table 4 Relative 15N-specific allocation (RSA) and RSAgrain:RSAwhole-plant ratio in different maize (Zea mays) genotypes grown at low and high
nitrogen (N) fertilization inputs in 2001 following 15N labelling at the beginning of stem elongation
RSA at
silking (%)
RSAstover at
maturity (%)
RSAgrain at
maturity (%)
RSAwhole-plant
at maturity (%)
RSAgrain:RSAwhole-plant
at maturity
Genotype
Low N
High N
Low N
High N
Low N
High N
Low N
High N
Low N
High N
Déa
F2
Io
Mean
Standard errora
1.39
1.42
1.58
1.46
1.11
1.16
0.73
0.98
0.96
0.80
0.82
0.86
0.84
0.84
0.76
0.86
0.98
0.79
0.73
0.83
0.71
0.81
0.67
0.73
0.97
0.79
0.77
0.84
0.75
0.82
0.71
0.76
1.00
1.00
0.95
0.98
0.94
0.98
0.95
0.96
0.12
0.15
0.14
0.14
0.01
a
Standard error for a genotype mean.
Fig. 2 Relationship between the relative 15N-specific allocation for
the grain (RSAgrain) and that for the stover (RSAstover) at maturity in
the 2001 experiment following labelling during stem elongation in
maize (Zea mays). There were 18 observations, including three per
combination of genotype (Déa, Io and F2) × nitrogen level (N0 or N1).
analysis of variance, thus leading to nonsignificant genotype,
N fertilization, and genotype × N fertilization effects (Tables 2, 3, 4).
In contrast, when considering the RSAgrain:RSAwhole-plant
ratio, a low standard error and a low coefficient of variation
(Tables 3, 4) were obtained with a highly significant genotype
effect in 2001 and an effect at the limit of significance in
2002. Furthermore, in the 2001 experiment the influence of
the level of N fertilization on the RSAgrain:RSAwhole-plant ratio
was highly significant (0.96 at high N input and 0.98 at low
N input; Tables 2, 4); the interaction genotype × N fertilization level was only significant at the probability 0.058. When
the RSAgrain:RSAstover ratio was considered, it was also found
to be estimated with higher accuracy than individual RSAs,
but not as accurately as the RSAgrain:RSAwhole-plant ratio
(coefficients of variation of 4.8% and 5.6%, compared with
1.9% and 1.8%, respectively, in 2001 and 2002).
A close relationship between RSAstover and RSAgrain was
systematically observed whatever the year of experiment. The
correlation between the two parameters was 0.96*** in 2001
(18 data points), 0.95*** in 2002 (16 data points) and 0.94***
when the 2001 and 2002 data were pooled (Figs 2, 3). The
regression coefficient, for a regression line with zero
intercept, was 0.94 in 2001 and 1.04 in 2002. The RSAgrain:
RSAstover ratio was therefore always close to 1 (Tables 3, 4).
Similarly, the RSAgrain:RSAwhole-plant ratio was always near 1,
ranging between 0.95 for the genotype Io in 2001 and 1.03
for the commercial hybrid Nicco in 2002. Consequently,
a regression coefficient close to 1 was obtained for the relationship between RSAgrain and RSAwhole-plant by using the 34
data points of both years (Fig. 3), with a strong phenotypic
correlation (0.99***) between RSAgrain and RSAwhole-plant.
At 35 DAS in 2001 and 2002, the analysis of variance
(ANOVA) showed that the results were similar to those
obtained at maturity, with a large coefficient of variation
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New Phytologist (2006) 172: 696– 707
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702 Research
Table 6 Relative 15N-specific allocation (RSA) values and ratios at
different stages of maize (Zea mays) plant development in 2002
following 15N labelling just after silking
Fig. 3 Relationship between the relative 15N-specific allocation for
the grain (RSAgrain) and that for the stover (RSAstover) following
labelling at the beginning of stem elongation in 2001 and 2002 and
harvesting at maturity and 35 d after silking in maize (Zea mays).
leading to no significant genotype effects for RSAs and a
lower coefficient of variation for the ratios RSAgrain:RSAstover
and RSAgrain:RSAwhole-plant than with individual RSA values.
However, the genotype effect for these ratios was significant
(Table 5). In the 2002 experiment, the regression coefficient
of RSAgrain onto RSAstover was close to 1 (0.99) at 35 DAS
with a correlation coefficient equal to 0.94***. Consequently,
the regression coefficient for RSAgrain onto RSAwhole-plant was
again close to 1 (Fig. 3). This means that the quasi-equality to
1 observed for the ratio RSAgrain:RSAwhole-plant already existed
before maturity when N remobilization and N uptake were
still active. Only in the 2001 experiment was the slope of the
regression of RSAgrain onto RSAwhole-plant (0.85) significantly
lower than 1 (Table 5).
Labelling after silking When 15N labelling was applied just
after silking, the genotype effect was not significant for
RSAgrain, RSAwhole-plant and the RSAgrain:RSAwhole-plant ratio,
whatever the sampling date (15, 25 or 35 DAS or at maturity).
Nevertheless, the coefficient of variation was lower for the
RSAgrain:RSAwhole-plant ratio than for RSAgrain and RSAwholeplant individually (Table 6). The regression coefficients (b) of
RSAgrain onto RSAstover were always associated with a high
correlation coefficient (varying from 0.77** to 0.96**). They
decreased significantly from 15 DAS (b = 2.71) to maturity
RSAstover
Mean (%)
Coefficient of
variation
RSAgrain
Mean (%)
Coefficient of
variation
RSAgrain:RSAstover
Mean
Coefficient of
variation
RSAgrain:RSAwhole-plant
Mean
Coefficient of
variation
Correlation RSAgrain
with RSAstover
Regression RSAgrain
onto RSAstover
Slopea
Intercept
15 DAS
25 DAS
35 DAS
Maturity
0.66
22.6
0.67
19.9
0.61
20.2
0.52
24.5
1.83
26.8
1.71
22.3
1.53
16.4
1.24
16.2
2.79
8.3
2.56
11.6
2.53
9.1
2.44
11.7
2.28
6.4
1.85
4.8
1.54
3.7
1.23
3.0
0.96***
0.85***
0.91***
0.90***
2.71A
0.03 (ns)
2.25AB
0.19 (ns)
1.69BC
0.49*
1.57C
0.43*
Significance: *, P < 0.05; ***, P < 0.001; ns, not significant.
a
Two values with the same superscript letter (A, B or C) are not
significantly different at P < 0.05.
DAS, days after silking.
(b = 1.57). Regression of RSAgrain on RSAwhole-plant gave
lower slopes: b = 2.2 for 15 DAS and b = 1.2 at maturity with
a zero intercept (Fig. 4), whereas the associated correlation
coefficients were higher.
The same results were obtained using the data from the
2004 experiment. Pooling the six observations (two genotypes
and three replicates) at a given stage of plant development, the
average ratio R = RSAgrain/RSAwhole-plant derived from the linear
regression of RSAgrain on RSAwhole-plant, with the constraint of
a zero intercept, was highest at 15 DAS (R = 1.44, r = 0.91**,
Table 5 Relative 15N-specific allocation (RSA), RSA ratios and relationships between RSAgrain and RSAstover or RSAwhole-plant in maize (Zea mays)
at 35 d after silking in 2001 [at low N input (N0) and high N input (N1)] and in 2002, following 15N labelling at the beginning of stem elongation
RSAg vs RSAst
2001, N0
2001, N1
2002
RSAstover
mean (%)
RSAgrain
mean (%)
RSAg:RSAst
mean
RSAg:RSAwp
mean
1.16 (25.7)a
0.90 (25.7)
0.87 (18.6)
1.01 (25.5)
0.76 (25.5)
0.86 (20.6)
0.87 (4.2)
0.84 (4.2)
0.98 (5.8)
0.84 (3.0)
0.80 (3.0)
0.99 (3.5)
RSAg vs RSAwp
Correlation
Regression
Correlation
Regression
0.96***
0.94b
0.96***
0.85b
0.94***
0.99b
0.98***
1.01b
a
Coefficient of variation; bwith zero intercept.
Significance: ***, P < 0.001.
RSAg, RSAgrain; RSAst, RSAstover; RSAwp, RSAwhole-plant.
New Phytologist (2006) 172: 696–707
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Research
Fig. 4 Relationship between the relative 15N-specific allocation for
the grain (RSAgrain) and that for the whole plant (RSAwhole-plant)
following 15N-labelling at silking and harvesting at different stages
after silking (2002 experiment) in maize (Zea mays). The lines with
different slopes (b) are the regression lines with the intercept
constrained to be equal to zero.
r being the coefficient of correlation). R progressively
decreased from 25 DAS (R = 1.23, r = 0.93**) to maturity
(R = 1.19, r = 0.88*), but it remained significantly higher
than 1. A nearly identical R-value (R = 1.20, r = 0.99**) was
found with data collected from an experiment performed on
100 different genotypes (data not shown) using the same
computing method (Coque, 2006).
Fig. 5 Simulation of the effect of 15N labelling during stem
elongation on the ratio RSAgrain:RSAwhole-plant (RSA, relative
15
N-specific allocation) based upon the amounts of nitrogen (N)
present in the whole plant and in the kernel at different stages of
plant development in the maize (Zea mays) hybrid Déa; (a) in 2001
and (b) in 2002.
Modelling
Following 15N labelling during stem elongation in 2001,
x = 0.55 (the proportion of the newly synthesized amino
acids originating from postsilking N uptake allocated to the
grain) yielded the best fit to the observed variation of
the RSAgrain:RSAwhole-plant ratio during the period spanning
35 DAS to maturity (Fig. 5a). In the 2002 experiment,
x-values of approx. 0.20–0.30 fitted more accurately the
observed results (Fig. 5b). For labelling after silking in the
2002 experiment, the model clearly predicts a decrease in
the RSAgrain:RSAwhole-plant ratio, as observed from silking to
maturity. This leads to x-values ranging between 0.30 and
0.55, with x-values increasing from 15 DAS to maturity
(Fig. 6).
Fig. 6 Simulation of the effect of 15N labelling at silking on the ratio
RSAgrain:RSAwhole-plant (RSA, relative 15N-specific allocation) based
upon the amounts of nitrogen (N) present in the whole plant and in
the kernel at different stages of plant development in the maize (Zea
mays) hybrid Déa in 2002.
Discussion
Explanation for the relationship between RSAgrain
and RSAwhole-plant
15N
labelling performed either before or after silking always
provided a close relationship between RSAgrain and RSAstover
or between RSAgrain and RSAwhole-plant, at all stages of
development, the latter relationship being slightly stronger
than the former because of the contribution of RSAgrain to
RSAwhole-plant. The proportionality between RSAstover and
RSAgrain at all times could have been caused either by an
exchange of N between the stover and the grain or by a
common source of variation for N allocation to the two
organs. It is logical to find that, for 15N labelling at the
beginning of stem elongation, the isotope was also allocated
to the grain, as a result of remobilization. However, N
remobilization alone cannot explain the finding that RSAstover
was very similar to RSAgrain. For 15N labelling after silking, the
situation is easier to explain because the 15N tracer appeared
to be simultaneously distributed to the stover and to the grain.
Nevertheless, neither the linear relationship observed between
© The Authors (2006). Journal compilation © New Phytologist (2006) www.newphytologist.org
New Phytologist (2006) 172: 696– 707
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RSAgrain and RSAstover nor the decrease towards 1.0 of the
RSAgrain:RSAwhole-plant ratio from 15 DAS to maturity can be
readily explained by 15N distribution between the stover and
the grain.
For 15N labelling at the beginning of stem elongation, the
strong relationship found between RSAgrain and RSAstover
could be a result of significant 15N absorption after silking. In
the 2001 and 2002 experiments, the influence of the genotype
on postsilking 15N uptake was not significant because of a
large environmental effect. On average, in 2001 there were N
losses (9%) between silking and maturity, but they were not
significant. Therefore, for this year, one can assume that 15N
uptake after silking was practically absent. In 2002, on average
23.1% of the 15N present in the whole plant at maturity was
taken up after silking. However, this difference was just at the
limit of significance at the threshold of 0.05 (data not shown).
Even if the contribution of 15N uptake after silking was considered to be significant in 2002, it is not sufficient to explain
the strong relationship between RSAgrain and RSAwhole-plant
also observed in 2001 in the absence 15N uptake after silking.
Whatever the type of labelling, another important factor
explaining the relationship observed between RSAstover and
RSAgrain is their large environmental variation, which
reflected the large environmental variation in the amount of
labelled fertilizer taken up by each plant under field growth conditions. Indeed, in the absence of isotopic discrimination, when
environmental variations affect RSAstover they are expected
also to affect RSAgrain in the same proportion. Considering
only the environmental correlation between RSAgrain and
RSAstover, a correlation of 1 may be expected between the two
RSA values. Therefore, it appears that the RSAgrain:RSAstover
and RSAgrain:RSAwhole-plant ratios are key parameters whatever the plant developmental stage because: (1) they provide
a good picture of 15N distribution between the grain and the
stover; (2) they can be accurately determined as they are not
affected by environmental variation such as 15N availability;
and (3) the RSAX:RSAwhole-plant ratio can be used to estimate
the rate of N allocation to a particular organ X (Cliquet et al.,
1990a,b; Deléens et al., 1994).
It is now necessary to explain why the regression coefficients for
both RSAgrain onto RSAstover and RSAgrain onto RSAwhole-plant
were always close to 1 from 35 DAS to maturity following
15N labelling at the beginning of stem elongation, whereas for
15N labelling at silking the coefficients were both much higher
than 1 and tended to decrease towards 1 from 15 DAS to
maturity. It is likely that such changes in the two RSA ratios
indicate a movement towards an equilibrium corresponding
to an equivalence between RSAgrain and RSAstover, whatever
the period of 15N application. With labelling at the beginning
of stem elongation, assuming that N remobilization and N
uptake occur simultaneously, postsilking allocation to the
stover of the N taken up by the plant will dilute the 15N
already present in the stover, while the grain will be enriched
in 15N by remobilization from the stover. Conversely, when the
New Phytologist (2006) 172: 696–707
15N
fertilizer is provided just after silking, N remobilization
from the stover to the grain will dilute the 15N allocated to the
grain, while at the same time the newly taken up N will be
allocated to the stover, thus enriching this organ with 15N.
Therefore, such N fluxes contribute to the progression of the
15N:14N isotopic ratio towards similar values in the stover and
in the grain.
Considered overall, the proposed model provides a good
simulation, fitting the results obtained in the field experiments. In the 2001 and 2002 experiments and with the two
types of labelling, there was an allocation of newly synthesized
amino acids to the stover (1 − x) ranging between 45 and
70%. The model suggests the hypothesis that, after silking,
the amino acids from the newly assimilated N are in part
mixed with amino acids from proteolysis for the synthesis
of new proteins which are further hydrolysed (Fig. 1). This
mixture modifies the RSAgrain:RSAwhole-plant ratio during grain
filling in such a way that it reaches a final value close to 1.
However, the situation for which RSAgrain ∼ RSAstover is easier
to reach when labelling during vegetative growth is used, rather
than labelling at silking. Indeed, with labelling during vegetative
growth, in the absence of N uptake after silking, RSAgrain
would strictly be equal to RSAstover. When there is postsilking
N uptake and allocation of N originating from the newly synthesized protein to the stover and to the grain, a dilution effect
will occur. Therefore, the ratio RSAgrain:RSAwhole-plant will be
below 1 at the beginning of grain filling, but thereafter will
rapidly increase towards 1. The fact that grain filling begins
actively only 10–15 d after ovule fertilization can also quickly
lead to a RSAgrain:RSAwhole-plant ratio close to 1. Indeed, at the
end of this period, whole-plant N represents c. 85% of the
whole-plant N at maturity and c. 70% of the N in the grain.
Therefore, the contribution of N uptake will be relatively
small in comparison to that of remobilization from the stover
to the grain and RSAgrain will be close to RSAstover. When
considering labelling at silking, even with a mixture of new
(synthesized following N uptake) and pre-existing amino
acids (originating from remobilization), RSAgrain will be
much higher than RSAstover. Consequently, at the beginning
of grain filling, the RSAgrain:RSAstover ratio will be greater than
1, but will decrease towards 1 as a result of a dilution effect
occurring in both the stover and the grain. This was verified
by our simulation model, which showed an increase in the
proportion of newly synthesized amino acids allocated to the
kernels (x) from 15 DAS to maturity. From a physiological
point of view, it is likely that this proportion increases with
time, while leaf senescence is progressing, as already shown by
Weiland (1989) and Ta & Weiland (1992) in maize and by
Schiltz et al. (2005) in pea.
The allocation of newly synthesized amino acids to the
stover must be mainly related to the leaf protein turnover
occurring in the stover (Hirel & Gallais, 2006). As there is no
increase in the amount of protein in the stover, this allocation
is probably occurring to replace the old proteins that have
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Research
been remobilized. By simulation, we found that a protein
lifetime of c. 5 d is sufficient to generate the equivalent of a
complete mixture of amino acids from newly assimilated
N with amino acids from proteolysis. In fact, our model
corresponds to a very quick protein turnover. This finding is
consistent with the results obtained by Lattanzi et al. (2005),
who showed that during vegetative growth most of the N
imported into growing tissue originated from storage pools.
Moreover, Liu & Jagendorf (1984), and Malek et al. (1984)
showed that, in pea, 20–35% of the proteins newly synthesized by isolated chloroplasts were degraded during the next
30 min. Therefore, the agreement between our model and the
experimental results suggests that, after silking, the proteolysis
of ageing proteins provides a pool of amino acids which can
be either translocated to the grain or recycled for the synthesis
of new proteins from a mixture which also contains amino
acids recently synthesized following N uptake. Our model is
thus close to that proposed by Cooper & Clarkson (1989), in
which the amino acids that are cycling between shoots and
roots are conceived as a single pool.
Validity of the different assumptions
For 15N labelling at the beginning of stem elongation, it was
assumed that 15N uptake after silking is negligible. We have
already discussed that the impact of 15N uptake after silking,
which was observed in 2002 but not in 2001, was expected to
be low. However, experiments are now in progress to evaluate
whether it is possible to reduce the risk of significant residual
15N uptake after silking by providing 15N much earlier, i.e.
before stem elongation. We expect to find that the kinetics of
15N uptake are similar to that observed by Sheehy et al.
(2004a,b), who showed that in rice 90% of the 15N was taken
up 20 d after labelling.
The second assumption required for the validity of our
experimental design is that 15N is homogeneously distributed
within the plant, proportionally to the amount of N in each
organ, and that isotopic equilibrium is reached. Isotopic equilibrium is favoured by the time interval between labelling and
silking, and also by the low amount of 15N provided per plant.
Redistribution of absorbed N among the leaves, as suggested
by the results of Simpson et al. (1983) in wheat and those of
Schiltz et al. (2005) in pea, would contribute to fulfilling the
assumption of homogeneous N distribution among vegetative
organs. Similarly, Lattanzi et al. (2005) showed that N tracer
entered into storage pools more than once before reaching the
growth zone, which is in favour of a homogeneous distribution. An exchange between the phloem and the xylem
could also favour homogeneous 15N distribution within the
plant (Cooper & Clarkson, 1989). It was in order to favour
such a homogeneous distribution of the isotope that we chose
to apply 15N just before stem elongation, which corresponds
to the beginning of the high growth rate period. If there is a
nonhomogeneous N distribution among vegetative organs,
but if the same proportion of N is remobilized from the
different parts of the plant, the conclusions derived from our
model are still valid (see Appendix). Furthermore, the simulation of observed results in our model is a proof that such an
assumption tended to be satisfied.
The third assumption in our work concerns the absence of
14N and 15N discrimination by the plant for N distribution
and N remobilization. RSAs were higher at low N input than
at high N input, suggesting that discrimination may have
occurred either at the level of N uptake or during the first
steps of inorganic N assimilation (Mariotti et al., 1982;
Ledgard et al., 1985; Coque et al., 2005). However, such
discrimination does not have any effect on the RSA ratio,
because the RSA for each organ will be modified in the same
proportion. Only differential discrimination for the synthesis
of new amino acids originating from N uptake and for amino
acids released following protein hydrolysis could affect the result.
Another possible bias could be introduced if there are N losses
as a result of NH3 emission during senescence (Schjoerring,
1991; Sharpe & Harper, 1997). However, if there is no
discrimination and if there is an equal distribution of the two
isotopes among vegetative organs, the RSA value of the stover
will not be affected as it is dependent on the 15N:14N ratio. If
the stover is considered as a sufficiently homogeneous organ
for N distribution, the ratio RSAgrain:RSAstover will not be
affected by the losses, whereas the ratio RSAgrain:RSAwhole-plant
will be. However, the bias introduced by N losses is expected
to be generally low (see Appendix). Furthermore, if N losses
did take place, as is probable in the 2001 experiment, it does
not appear that they affected either the relationship between
RSAgrain and RSAstover or the predictive value of our model.
For a labelling treatment just after silking, the most important assumption is that 15N is homogeneously distributed in
the different soil compartments colonized by the roots during
the entire period of postsilking N uptake. As discussed before,
if new amino acids originating from the N recently taken up
are distributed in the different parts of the plant, the heterogeneous N distribution in the soil will not have any significant
impact. Concerning N availability during the time span from
flowering to maturity, it must be noted that N availability in
the first 30 d after silking, during which 90% of the total
plant N is absorbed, will be the most important. One can
hypothesize that, if the availability of 15N in the soil decreases
with time, this will tend to enhance the decrease of RSAgrain
during the same period. However, the simulation of observed
results by our model showing a decrease in the ratio
RSAgrain:RSAwhole-plant from 15 DAS to maturity is a proof
that the assumption of a homogeneous 15N distribution in the
different soil compartments colonized by the roots tended to
be satisfied, at least at the most important phase of postsilking
N uptake. Nevertheless, as a whole, the assumptions required
for reliable interpretation of the results of our 15N-labelling
experiment appear to be more difficult to fulfil for labelling after
silking than for labelling at the beginning of stem elongation.
© The Authors (2006). Journal compilation © New Phytologist (2006) www.newphytologist.org
New Phytologist (2006) 172: 696– 707
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However, the value of using 15N labelling after silking was
to show that there is direct allocation of N to the stover,
originating from postsilking N uptake.
Conclusion
The use of 15N labelling at the beginning of stem elongation
and just after silking allowed the demonstration that, in maize,
a large proportion (> 50%) of newly synthesized amino acids
from postsilking N uptake were first integrated into the stover
proteins before translocation to the grain. This finding indicates
that protein turnover, which is controlled by an intra-organ
regulatory mechanism, is occurring concomitantly with N
remobilization to the grain, which represents an inter-organ
metabolic process. It remains to be determined whether the
proportion of N allocated to the stover and the proportion of
N remobilized have a direct link to the protein turnover
occurring at the cellular level (Brouquisse et al., 2001;
Hörtensteiner & Feller, 2002). The occurrence of such a link
would mean that the proteolytic enzymes involved in the
control of protein turnover could also be involved in the
degradation of leaf protein during senescence. Mae et al.
(1983), Huffaker (1990) and Hörtensteiner & Feller (2002)
suggested that certain common catabolic enzymes may
function during the initial and reversible phase of senescence,
while others are specifically induced when senescence is
progressing. Therefore, the occurrence of some common and/
or distinct mechanisms regulating protein turnover and
protein hydrolysis during leaf N remobilization (Irving &
Robinson, 2006) must be taken into consideration during the
general process of plant N management.
Acknowledgements
The authors are very grateful to Dr Peter Lea for his careful
revision of the English. We also thank very much Dr Gilles
Lemaire (INRA, Lusignan, France) and Dr Jacques Le Gouis
(INRA, Mons-en-Chaussée, France) for their helpful discussions
about the experimental results and the model.
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Appendix: Discussion of Some Assumptions for
Labelling Performed at the Beginning of Stem
Elongation
(1) Homogeneity of 15N:14N ratio in the stover
The model assumes the existence of a stover compartment
with a homogeneous 15N distribution. In fact, as demonstrated
below, the distribution can be heterogeneous among vegetative
organs if the same proportion of N is remobilized to the grain
in each organ.
Let T1 and T2 be two stover organs, with Q1 and Q2 total
amount of N and q1 and q2 the amount of 15N, respectively,
and assuming that the 15N abundance in each organ q1/Q1 is
different from q2/Q2. Let g1 and g2 be the 15N quantity in the
grain originating from T1 and T2, respectively, and p1 (p2) the 15N
amount at silking in the organ. Then, the proportion remobilized
from each organ is r1 = g1/p1 and r2 = g2/p2. Now if we assume
that the two organs remobilize the same proportion of their N:
r1 = r2 = g1/p1 = g2/p2 = (g1 + g2)/(p1 + p2) = Σi gi/Σi pi = g/wp
(with i = 1, 2)
(g, the 15N quantity in the grain; wp, the total 15N amount in
the whole plant at maturity.)
Therefore, with the assumption of the same proportion
remobilized from each organ, despite the heterogeneous
distribution of N, the different vegetative compartments can
be pooled, which is equivalent to assuming a homogeneous
distribution of 15N in the different organs. It is only with a
heterogeneous 15N distribution and heterogeneous proportions remobilized among vegetative organs that the estimation
of the proportion of remobilized N will be biased.
(2) Effect of N losses on the relationship between RSAs
for labelling at the beginning of stem elongation
Consider that the stover (S) consists of leaves (L) and stalks (T),
and let RSAL, RSAT and RSAS be the RSAs for leaves, stalks
and stover, respectively. QL and QT being the total amount of
N in leaves and stalks, without N losses, the stover RSA is:
RSA S =
RSA LQ L + RSA TQ T
.
QL + Q T
Assuming that there are losses only affecting the leaves, these
losses being Qp, then the new stover RSA, estimated at
maturity, will be
RSA ′S =
RSA L (Q L − Q p ) + RSA TQ T
QL + Q T − Qp
which can be reformulated as
RSA ′S = RSA S
1 − pR
1− p
[p = Qp/(QL + QT); R = RSAL/RSAT.]
As an example, if R = 1.20 and p = 0.15 (15% N losses),
then RSA′S = 0.96 RSAS. This means there is a low bias. Thus
it would be only with large and unlikely losses that the RSAS
would be significantly affected.
© The Authors (2006). Journal compilation © New Phytologist (2006) www.newphytologist.org
New Phytologist (2006) 172: 696– 707
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