108
2.5 The effects of ammonium nutrition, N supply and internal N remobilization
on 15N signatures of genotypes of F. rubra and A. capillaris
2.5.1 Introduction
Ammonium is the major inorganic form of N available to plants in upland pastures like
the field sites used in this project. During the course of a year in which soil from Cleish
was sampled and extracted biweekly, ammonium was the only measurable form of
inorganic N (data not shown). Similar observations were also made for two other
grasslands in Scotland (Williams 1999). Ammonium nutrition, however, is known to
affect plant 15N signatures differently to nitrate nutrition. Characteristic are distinct
whole plant discriminations and a lack of discrimination among different plant parts
(Yoneyama et al. 1991). The previous chapters (2.1 –2.3) have shown that N derived
from remobilization from storage pools or through recycling can contribute
substantially to the N found in a new tissue. The 15N value of remobilized N is not
known, but if it was isotopically distinct from the 15N value of currently assimilated N,
could provide a tool for measuring N remobilization in the field. The experiments
described here want to determine (1) the influence of genotype and ammonium nutrition
under two N supply rates on 15N discrimination of the whole plant and its parts, and (2)
the 15N signature of remobilized N.
2.5.2 Materials and Methods
Plant material, growing conditions and harvests
The plant material from the first harvest from the second labelling experiment (see
chapter 2.3) was analysed for its 15N signatures. Briefly, five F. rubra and A. capillaris
genotypes were grown in sand culture in a controlled environment room under low
(0.06 mmol N week-1) or high (1 mmol N week-1) N nutrition, with ammonium being
109
the only N source. The nutrient solution was freshly prepared from concentrated stock
solutions for each feeding. The ammonium sulphate stock solution was frozen in small
aliquots; once defrosted, it was kept at 4C and used for a maximum of two weeks.
After potting, plants were grown for 20 days to allow establishment, and a further 46
days under “summer” conditions, then destructively harvested and separated into the
youngest (1st) leaf, the remaining living leaves, dead leaves and roots.
For two genotypes of each species (genotypes 1, 2, 6 and 7), two additional harvests
were taken. These later harvests correspond to the early and intermediate spring
harvests (H4 and H5) of the labelling experiment, 150 and 190 days after the first
harvest. The plants receiving unenriched nutrient solution were grown under identical
conditions to those from the tracer study, apart from being kept in a separate growth
room to avoid tracer
15
N contamination. In the same way as the
15
N-labelled plants, a
subset of tillers had been marked at the start of the particular season with paint in order
to distinguish between old and new growth. At harvest, shoot material was separated
into dead leaves, old living leaves (OL) and new living leaves (NL). The latter two
combined gave living leaves (LL). New leaves at H4 or H5 were those that had grown
since H3 or H4, respectively. Living leaves at H4 (LL4) still living at H5 would then be
old leaves (OL5). Root material was not sampled, and dead leaves were not analysed for
their isotopic signature. Isotope analysis of oven-dried material was as in the experiment
with the five A. capillaris genotypes (section 2.4).
Calculation of 15N signatures of remobilized N and N derived from uptake
For the harvest interval 4  5 changes in the 15N signatures were compared with data
obtained from the labelling experiment for the same genotypes. The equations below
were used to calculate the 15N signatures of leaf N taken up during this interval, and
the 15N signatures of internal N translocated into new leaves and those of internal N
110
remaining in old leaves after translocation. 15N values were derived from the
unlabelled plants, N pool sizes of internal, uptake N and total N (intN, uptN, totN) from
the labelled plants (see Fig. 16). Calculations were performed for the four genotypes,
however only the mean values of those four genotypes are presented. Changes in 15N
and N content of living leaves between H4 and H5 are due to uptake of N (as no major
fluxes of internal N from the root to shoot or vice versa occurred). It was assumed that
the uptake N in old and new leaves had identical 15N signatures.
NL5
15intNNL5
?
[uptN]NL5
[intN]NL5
15uptN
?
[totN]LL4
15totNLL4
LL4
LL5
[totN]LL5
[intN]OL5
15totNLL5
 intNOL5
?
15
[uptN]OL5
OL5
Fig. 16: N pools and their isotopic signatures at H4 and H5. Variables in boxes are derived from
the labeling and natural abundance experiments. N present in living leaves at H4 (represented
by black and white stripes) either remains in these leaves (then designated old leaves) or is
translocated to new leaves. N taken up between H4 and H5 (represented by grey shade) is
allocated to both old and new leaves.
15totNLL(5) * [totN]LL(5) - 15totNLL(4) * [totN]LL(4)
(1) 15uptNLL(5) =
[uptN]LL(5)
15totNNL(5) * [totN]NL(5) - 15uptNLL(5) * [uptN]NL(5)
(2)  intNNL(5) =
15
[intN]NL(5)
15totNNL(5) * [totN]OL(5) - 15uptNLL(5) * [uptN]OL(5)
(3)  intNOL(5) =
15
[intN]OL(5)
111
Statistical analysis
At H1, data was analysed with ANOVA to test for the effect of species, genotype
(nested within species) and treatment and, if applicable, plant part, and the interactions
of those main effects. When tested for plant parts, the block structure was plant number.
For statistical analysis of data from the four selected genotypes, main effects were
genotype, treatment and harvest (and plant part).
2.5.3 Results
15N signatures of F. rubra and A. capillaris genotypes under ammonium nutrition
N source and whole plant 15N signatures
The 15N value of the ammonium in the nutrient solution was 4.7‰. On average, plants
were depleted in
15
N relative to the nutrient solution by 2.1‰. An exception was
genotype 6 that in the LN treatment had a whole plant signature identical to the nutrient
solution. The genotypic range of whole plant 15N values in F. rubra and A. capillaris
genotypes was 1.2‰ and 2.4‰ for LN plants, and 0.4‰ and 0.7‰ for HN plants.
The effects of treatment, species and genotypes on 15N values
Table 15: P values of ANOVA testing 15N values of whole plants, different plant parts
and differences between parts at the first harvest.
species (s)
treatment
(t)
genotype
(g)
sxt
gxt
0.398
0.003
living
leaves
0.843
0.132
1st leaves dead
leaves
0.194
0.475
<0.001 0.296
0.116
0.017
0.062
<0.001
0.117
<0.001
0.003
<0.001
0.007
0.004
0.010
<0.001
0.012
0.010
0.050
whole
plant
0.298
0.337
root
112
Table 15 summarises the ANOVA results for 15N signatures of whole plants and their
different organs. The species had no significant effect on the 15N value of either whole
plants or their different parts. For whole plants, living leaves (1st leaves not included)
and dead leaves neither treatment nor genotype affected 15N values. The root system
was more enriched in
15
N under LN as compared to HN, and genotype significantly
influenced root 15N values. Genotype as well as treatment affected the 1st leaf 15N
values, with LN resulting in lower 15N values than HN.
Plant organ 15N
8
7
LN
6
5
4
3
15N (‰)
2
1
HN
root
7
living leaves
6
1st leaves
5
4
3
2
1
0
1
2
3
4
5
6
F. rubra
7
8
9
10
A. capillaris
genotypes
Fig. 17: 15N values of 1st leaves, living leaves and roots of 5 genotypes of F. rubra and A.
capillaris grown under low or high N supply at H1. Shown is the average of 3 replicates and the
standard errors if they exceed the size of the symbol.
113
Fig. 17 shows the 15N values for 1st leaves, living leaves and roots for each genotype
under LN and HN. Dead leaves are not shown as their N content was small and their
15N values were not significantly different from those of living leaves (see Table 16).
Under LN, 1st leaves were more depleted in 15N than living leaves (except genotype 1),
whereas under HN that pattern was reversed (except genotypes 5 and 8, which were
depleted and genotype 10, which had identical values for both leaf categories). Table 16
shows that the significant differences between living leaves and 1st leaves are controlled
by treatment and genotype only.
Table 16: P values of ANOVA testing for differences in 15N values
between parts at H1. Comparisons were for dead leaves versus living
leaves, 1st leaves versus living leaves and the total of living leaves
(including 1st leaves) versus roots.
part (p)
p x species (s)
p x treatment (t)
p x genotype (g)
pxsxt
pxgxt
dead leaves,
living leaves
0.238
0.464
0.871
0.052
0.221
0.111
1st leaves,
living leaves
0.042
0.142
<0.001
0.001
0.947
0.174
living leaves+1st,
roots
0.004
0.338
<0.001
0.024
0.216
0.339
Significantly different enrichments occurred also between roots and living leaves due to
effects of treatment and genotype (Table 16). The comparison is made for the total of
living leaves, which is the sum of living leaves and 1st leaves (living leaves+1st). The
15N values of this combined fraction is very close to that of living leaves, as the N
content of the 1st leaves was small. For LN conditions the living leaves+1st were depleted
in
15
N relative to the root system (except genotype 5), whereas for HN the majority of
the genotypes (apart from genotypes 2 and 4) were enriched in 15N in the leaves relative
to the roots.
114
Within a plant, the difference among different plant parts was on average higher in
plants grown under LN conditions than in plants grown under HN. For LN, this
difference between roots and living leaves+1st was 1.0‰ and 1.9‰ for F. rubra and A.
capillaris genotypes, and for HN, it was 0.4‰ and 0.6‰, respectively.
15N signatures and N concentration of live shoot material over time
15N
N concentration
5
4
G1
G2
LN
4
G6
G7
LN
3
3
2
2
1
1
15N
(‰)
HN
N conc.
(% of dw)
HN
4
3
3
2
2
1
1
0
0
1
4
5
1
4
5
Harvest
Fig. 18: Mean 15N signatures and N concentration of the live shoot material of four genotypes
at three harvests representing summer (H1), and following early and late spring (H4 and 5).
Plants were grown either under low or high N supply. F. rubra genotypes are represented as
circles, A. capillaris genotypes as triangles.
The 15N signatures of the total of living leaves were observed for two genotypes of
both species at 3 points in time (see Fig. 18). These harvests represent the 1 st harvest of
the main experiment (summer; just described in detail for all genotypes) and 2 further
115
harvests corresponding to the two spring harvests (H4 and H5). The live shoot material
is a functional classification, as turnover and death of leaves had occurred over these
time periods. The harvest date had a significant impact (P < 0.001) on 15N signatures,
and this influence was dependent on treatment (harvest x treatment P < 0.001) and
genotype (harvest x genotype P = 0.012). Plants in the LN treatment were relatively
constant in their 15N values, whereas plants in the HN treatment – overall - declined in
15N between H1 and H4 and increased in 15N between H4 and H5. Also, under HN,
genotypes converged in their 15N signatures with time.
The feeding regime of the plants was a constant amount of nutrient solution with either
low or high N. Due to increases in plant size (tiller number and biomass) with time, the
constant addition would have resulted in a decrease of available N per tiller or biomass
present. The N concentration of the total of living leaves was used as an indicator for
the N status of the plant. For both low and high N conditions, the N concentration
changed with time, with the last harvest being significantly smaller than the two
preceding harvests (see Fig. 18).
15N signatures of new and old leaves
For H4 and H5, living leaves were separated into new and old live leaves. New leaves
had grown since the preceding harvest, with the average number of new leaves (on old
and new tillers combined) being 1.9 for LN and 2.2 for HN plants at both harvests dates.
The top panel in Fig. 19 depicts the allocation of total leaf N into new leaves. Strong
genotypic differences were evident. Overall, the patterns were very similar for both
harvests, enabling comparisons between them.
116
HN
LN
HN
LN
0.8
N allocation to NL
(fraction of NLL)
0.7
G1 G2 G6 G7
0.6
0.5
0.4
0.3
0.2
0.1
NS
4.5
4
15N (‰)
3.5
3
2.5
2
1.5
1
0.5
NL OL
0
H4
H5
Fig. 19: N allocation (top) and 15N values (bottom) at two harvests under low and high N
supply. In each box, mean values for genotypes 1 and 2 (F. rubra), 6 and 7 (A. capillaris) are
shown in this order from left to right. For 15N values, new and old leaves and the nutrient
solution (NS) are presented, whereas for the N allocation, the fraction of total living leaf N in
new leaves is given.
Across all harvests and treatments, new leaves had a 15N value significantly (see Table
17) below the 15N value of old leaves, and were therefore further away from the 15N
value of the nutrient solution. This isotopic separation between new and old leaves was
more pronounced at the later harvest. The difference between the two leaf fractions was
bigger in plants receiving LN nutrition as compared to HN.
117
Table 17: P values of ANOVA testing 15N values of new and old leaves
on marked tillers of four genotypes grown under low and high N and
harvested at H4 and H5. (genotype x trt x harvest x part; block = pot; not
all main effects and interactions shown)
part (NL,OL)
part x genotype
part x treatment
part x harvest
15N
<0.001
0.138
<0.001
0.058
Dynamics of 15N values for the growth period H4 to H5
In the labelling experiment (section 2.3) it was established that N losses (excluding N
bound in dead leaves) occurred from old leaves (LL4) due to transfer into new leaves
(NL5). Internal N recovered in new leaves at H5 can be accounted for by remobilization
from old leaves by 100% (LN) or 85% (HN); therefore no major net import of internal
N from the root system occurred. N taken up during this period and transferred to the
shoot was recovered in new leaves (40% LN; 51% HN) and old leaves (18% LN;
22%HN). The contribution of internal N to the total N in the new leaves was 55% for
LN and 69% for HN plants. As already mentioned above, the N addition per tiller (or
growing point or biomass) at the later harvests was decreased due to increases in plant
size. This meant that at H5, the amount of N taken up in relation to the amount of N
present in the plant was higher under LN (0.36) than under HN (0.22), and the amount
of N taken up in relation to plant biomass was identical under both treatments. The
distinction between LN and HN might, therefore, be more with respect to the history of
the plants, which was manifested in different biomass and N allocation patterns. LN
plants had a R/S ratio of total N of 1.0, whereas HN plants had 0.5. These different pool
sizes should be considered, especially because remobilization to NLs is mainly
supported from shoot N, and – in combination with lower relative N uptake - might
118
explain why new leaves of HN plants have a higher contribution of internal N to the
total N.
6
LN
5
4
OL
OL
3
2
NL
1
15N
(‰)
intN
5
totN
uptN
HN
4
3
2
1
0
H4
H5
Fig. 20: Measured and calculated 15N values of leaf material and its N constituents for the
growth period H4 to H5. Asterices ( ) indicate measured values; others are calculated. Presented
are the mean values for genotypes 1, 2, 6 and 7. OL4 corresponds to LL4. The 15N value of the
nutrient solution was 4.7 ‰.
Fig. 20 shows the dynamics of 15N signatures in the shoot material. The 15N values of
both new and old leaves at H5 will be determined by the 15N values of the internal N
and N taken up during the growth interval. Internal N present in old leaves at H5 had a
higher 15N value than at H4, whereas the N fraction remobilized from old leaves to
new leaves was more depleted. This depletion of translocated N was 0.5‰ or 0.4‰ for
LN or HN, respectively, relative to its source. N derived from uptake differed markedly
between treatments, with 15N values at LN of 2.2‰ and at HN of 5.1‰. Therefore,
119
uptake N under LN conditions was depleted relative to its external source by 2.5‰,
whereas under HN conditions, it was enriched by 0.3‰.
2.5.4 Discussion
15N values of F. rubra and A. capillaris genotypes under ammonium nutrition
F. rubra and A. capillaris genotypes were depleted in
15
N relative to their ammonium
source, and by this behaved the same as ammonium-grown Oryza sativa plants
(Yoneyama et al. 1991). However, with an average value of 2.1 ‰ the depletion in the
grasses was smaller than in the O. sativa plants, which differed from their N source
between 4.4 and 11.9 ‰ after 47 days of growth, depending on the concentration of N
in the solution (low or high N, respectively). In the experiment with O. sativa, two
harvests were taken either after 32 or 47 days of growth at both low and high N supply.
Treatment differences on the plant enrichment were not apparent at the earlier harvest,
corresponding to the absence of a treatment effect on whole plant 15N values in the
grass genotypes. Short-term measurements (30-120 min) of the fractionation expressed
by O. sativa plants receiving ammonium, measured after transfer in the nutrient
solution, was also higher under HN conditions than LN conditions (Yoneyama et al.
2001).
A further experiment investigating 15N signatures under ammonium nutrition in higher
plants was conducted with tomato, Lycopersicon esculentum in hydroponics (Evans et
al. 1996). These plants did not differ in their 15N values from the N source; however
plants were - compared with nitrate-grown plants – very small, and probably under
suboptimal growth conditions (with 50 mmol m-3 very high ammonium concentration in
the nutrient solution). It is interesting to note that genotype 6, which grew very poorly
under low N conditions, also had a 15N value very close to the N source.
120
For ammonium-grown plants previous reports showed no differences in 15N among
different parts (Yoneyama et al. 1991, Evans et al. 1996). The site of primary
ammonium assimilation is predominantely in the root, which supplies both root and
shoot with organic N, and therefore could result in similar 15N values. In this
experiment, an internal gradient of within-plant enrichments was observed. This
gradient (roots  bulk of living leaves  youngest leaves) was maintained under both N
regimes, though the direction of the gradient was opposite for the treatments, and its
magnitude bigger for the low N treatment. It is difficult to imagine a single mechanism
that would lead to this pattern. Possibly the identical whole plant enrichments of both
treatments are accidental, masking a very different nitrogen metabolism under the two
growing conditions. Variations between LN and HN plants could be possible in various
ways: (1) Preassimilatory efflux of
15
N-enriched ammonium could be responsible for
whole plant depletion. However, as shown by Yoneyama et al. (2001), this whole plant
discrimination could potentially be more expressed under HN than LN conditions. (2)
Transported N might have a 15N signature different to the N remaining in the tissue. If
this transport N was depleted relative to its source (see below), shoots would be
expected to be more
15
N-depleted than roots. This could explain the patterns seen for
LN plants. Furthermore, with increasing proportions of total N being moved from roots
to shoots, differences could be expected to become smaller. As HN plants have 70% of
their N content in the shoots, whereas LN plants have only translocated 53%, this could
potentially explain differences in the magnitude of differential enrichments. However, it
still does not justify that HN plants have
15
N-depleted roots. (3) Different transport
compounds with different enrichments could be used in HN plants as opposed to LN
plants. However, the phloem from leaves of Triticum aestivum did not differ with
respect to the amino acid composition in response to N supply (Caputo & Barneix
1997). (4) As discussed for the A. capillaris experiment, organic N losses could
121
influence 15N and Shoot-Root 15N signatures. This requires qualitative and
quantitative data on the effects of N supply on exudation of organic N and root tissue
turnover. (5) Increased ammonium translocation to the shoot with increasing N supply
(Finnemann & Schjoerring 1999) and its primary assimilation there could result in
higher 15N values in the shoots, as preassimilatory efflux of
15
N-enriched ammonium
as in the roots is not possible. (6) Increased ammonia emission from the shoot with
increasing N supply, which would, due to the likely 15N-depletion of lost ammonia, lead
to enrichment of the shoot (as seen under HN). Whether ammonia emission is important
in the studied grass species is not clear. (7) Grasses are well known for extensive shootto-root cycling of amino acid N (e.g. Simpson et al. 1982). An increase in the
magnitude of this process, as seen under higher external N concentrations (Agrell et al.
1994), could be responsible for the more homogeneous distribution of
15
N between
different plant parts under HN.
The amount of genotypic variation for 15N values was smaller than in those values
observed for five A. capillaris genotypes derived from the same field sampling and
studied under nitrate nutrition. Despite genotypic and species-specific differences in
other plant variables such as biomass, N content and their allocation (data not shown),
genotypic differences for 15N values were constrained to youngest leaves and roots and
the different enrichment patterns between plant parts. However, species or genotype x
environment interactions were significant in most instances, and confirm an observation
made in the A. capillaris experiment.
The range of whole plant 15N values was greater under LN conditions than under HN,
paralleling an observation made for Picea needles derived from either nitrate-poor or
nitrate-rich soils (Schulze et al. 1994). The variability in 15N across five A. capillaris
122
genotypes grown on nitrate also depended on the environmental conditions: stresses not
influencing plant N content (Low Light and Minus P) reduced the range of plant 15N as
compared to Control.
15N values of remobilized N
15N values measured in any plant part will depend on the 15N values of its two
principle N sources, N from current uptake and internal N. Differences in 15N values as
measured in this experiment between new and old leaves could be caused by (1)
differences in 15N values of N remobilized from, and N retained in, the source leaves
and/or (2) changes in the extent of discrimination during assimilation of newly absorbed
N.
Parallel experiments with
15
N labelled plants and plants at natural abundance provide
the opportunity of segregating measured total N 15N signatures into those of internal N
and uptake N. On this basis, it was deduced that N remobilized from old leaves (when
42% (LN) or 45% (HN) of initial internal N had been remobilized) was slightly
depleted relative to its source, regardless of treatment. Under LN, the 15N signature of
uptake N was similar to the 15N signature of internal N in new leaves. Under HN
however, uptake 15N values were very different to 15N values of leaves (and probably
the whole plant), and indicate that N discrimination has changed, probably due to an
increasing N limitation. Under field conditions, such a change could also be due to
changes in N source signatures. The fact that both changes in discrimination and N
sources can alter 15N values of uptake N constrains data interpretation of field
measurements.
Dead leaves were not analysed for their isotopic signatures at these later harvests,
however measurements from the first harvest as well as the A. capillaris experiment
123
(section 2.4) failed to detect significant differences in 15N between living and dead
leaves. Therefore, completed N translocation out of leaves is apparently without
discrimination. Incomplete remobilization as observed here is associated with
discrimination, with
15
N depleted N being preferentially remobilized from the
maturing/senescing leaves. The cytosolic form of glutamine synthetase is involved in
the generation of the transport amino acid glutamine (Edwards et al. 1990; Kamachi et
al. 1991), and could due to its discrimination against
15
N be responsible for the
observed depletion in 15N of remobilized N. An alternative explanation for dynamics in
signatures of remobilized N could lie in the orderly fashion of the senescence process
(Feller & Fischer 1994). The sequential breakdown of different cell compounds could
liberate differently enriched N at different times.
Comparisons made by Näsholm (1994) between the 15N values of green and yellow
(senescing) Pinus sylvestris needles, derived from field plots of varying fertility,
showed that the green needles were ~ 1‰ lower in 15N, irrespective of N supply. He
suggested that the enrichment in the yellow leaves was caused by volatilisation of
ammonia generated during senescence, however it could equally well be a consequence
of the mechanism described within this experiment. Also Kielland et al. (1998)
measured 15N of leaves collected at 3 different times in the year (corresponding to
young, mature and senesced leaf stages), finding no significant variation over the season
for four of the six species studied. In Picea mariana, however, young needles were
more depleted (1‰) than mature needles, whereas foliage of Populus tremuloides
(aspen), a species occurring at N rich sites, behaved the other way around. In that
respect there was a similarity to the measurements at the first harvest, where the
youngest leaves had lower 15N values than the older leaves under LN, but behaved
opposite under HN. It is known from the second harvest from the labelling study, that
LN plants relied more on N remobilization than HN plants (40% remobilized N in new
124
leaves under LN, 26% under HN). Potentially these lower values observed in the 1st
leaves under LN at H1 or the depleted P. mariana needles are an indication of higher
inputs from internal N sources.
2.5.5 Summary

Whole plant 15N was depleted in 15N relative to the N source by 2.1‰.

Whole plant 15N values in A. capillaris and F. rubra were not significantly affected
by N supply, and did not show genotypic differentiation.

Significant differences in 15N within a plant occurred between youngest leaf and
remainder of the living leaves, and between living leaves and roots. For LN plants,
roots had the highest and youngest leaves the lowest 15N, whereas HN plants
behaved opposite to that. These intra-plant differences were influenced by genotype,
and the range was higher under LN conditions as compared to HN.

15N values of the living leaf fraction changed with time.

When about half of the initial leaf N had been remobilized to the new leaves, the
15N value of N remobilized from leaves was slightly (0.4 – 0.5‰) depleted in 15N.