Journal of Experimental Botany, Vol. 57, No. 4, pp. 997–1006, 2006
doi:10.1093/jxb/erj085 Advance Access publication 17 February, 2006
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Impact of defoliation intensity and frequency on N uptake
and mobilization in Lolium perenne
F. Lestienne1, B. Thornton2 and F. Gastal1,*
1
Unité d’Ecophysiologie des Plantes Fourragères, INRA, Route de Saintes, F-86600 Lusignan, France
2
Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen AB15 8HQ, UK
Received 9 August 2005; Accepted 5 December 2005
Abstract
Introduction
The aim of the study was to evaluate the impact of
defoliation intensity, defoliation frequency, and interactions with N supply on N uptake, N mobilization
from and N allocation to roots, adult leaves, and growing leaves. Plants of Lolium perenne were grown under
two contrasted N regimes. Defoliation intensity treatments consisted of a range of percentage leaf area
removal (0, 25, 50, 75, or 100%). These treatments
were applied in parallel to a set of plants previously
undefoliated, and to a second set of plants which had
been defoliated several times at a constant height. A
15
N tracer technique was used to quantify N uptake,
mobilization, and allocation over a 7 d period. A significant reduction in plant N uptake was observed with
the removal of more than 75% of lamina area, but only
with high N supply. As defoliation intensity increased,
the amount of N taken up and subsequently allocated
to growing leaves during the labelling period was
maintained at the expense of N allocation to roots
and adult leaves. Increasing defoliation intensity increased the relative contribution of roots supplying
mobilized N to growing leaves and decreased the
relative contribution of adult leaves. Defoliation frequency did not substantially alter N uptake, mobilization, and allocation between roots, adult and growing
leaves on a plant basis. However, tiller number per
plant was largely increased under repeated defoliation,
hence indicating that allocation and mobilization of
N to growing leaves, on the basis of individual tillers,
was decreased by defoliation frequency.
Perennial grasses are often subjected to defoliation by
grazing animals. Defoliation intensity and frequency vary
to a large extent according to grazing and/or cutting management and according to animal type (Davies, 1988).
Grass plant physiology and, consequently, sward growth
and structure are highly affected by defoliation.
A single severe defoliation causes an immediate decrease in photosynthesis (Ludlow and Charles-Edwards,
1980; Parsons et al., 1983) and a reduction or cessation
in root growth (Davidson and Milthorpe, 1966; Ryle and
Powell, 1975; Jarvis and Macduff, 1989). Root (Oswalt
et al., 1959; Jarvis and Macduff, 1989) or even tiller (Gastal
and Saugier, 1986) senescence may occur after a single
severe defoliation. Respiration (Davidson and Milthorpe,
1966) and nutrient uptake (Clement et al., 1978) also
show a rapid decline after defoliation. Clement et al.
(1978) observed a strong relationship between NO
3 uptake
and CO2 influx in Lolium perenne after defoliation. As
photosynthesis and CO2 influx fell immediately following
defoliation, nitrogen uptake also declined. In such circumstances laminar regrowth is highly dependent on the plants’
capacity to mobilize and supply carbon and nitrogen to
growing leaves.
In the initial few days following defoliation, stubble
and roots reserves of C decrease (Davidson and Milthorpe,
1966; Gonzalez et al., 1989), also the amount of these
reserves mobilized to growing leaves increases (Ryle
and Powell, 1975). Additional studies (Prud’homme et al.,
1992; De Visser et al., 1997) demonstrated that such
mobilized reserves were indeed used by the growing
leaves. Analogous mechanisms exist for N, with N reserves
of both roots and shoots being mobilized to growing
leaves after defoliation (Ourry et al., 1988, 1989; Millard
et al., 1990; Lefevre et al., 1991). However, C or N
Key words: Defoliation intensity, defoliation frequency, Lolium
perenne, N mobilization, N uptake, N supply.
* To whom correspondence should be addressed. E-mail: [email protected]
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998 Lestienne et al.
reserves utilization may be insufficient to maintain leaf
growth at its pre-defoliation rate (Schäufele and Schnyder,
2000).
Many previous studies have concentrated on the effects
of a single severe defoliation upon grass physiology, but
fewer have addressed the effects of altering defoliation
intensity and/or frequency. A lower defoliation height was
shown to result in reduced N acquisition by four grass
species (Thornton and Millard, 1996). Repeated defoliation
allowed a substantial mobilization of N to growing leaves
in the study from Millard et al. (1990), but not in another
study from Thornton and Millard (1997), leaving open the
question of the impact of defoliation frequency on N
mobilization. Furthermore, the effect of defoliation on N
economy of grasses depends on their N nutrition. Plants
exposed to a low N supply showed a reduced downregulation of N uptake (Louahlia et al., 1999) and a reduced
mobilization of N (Thornton et al., 1993) in response to defoliation, compared with plants exposed to a high N supply.
Both Evans (1972) and Thornton and Millard (1996)
varied clipping height to achieve differences in defoliation
intensity. However, due to the fact that grass plants differ
in shoot morphology, in particular, between species or
during plant adaptation to frequent defoliation (Davies,
1988), clipping to a set height may remove differing proportions of the shoot material. Since at a whole plant
level N uptake is strongly related to the rate of photosynthesis, it would seem more pertinent to investigate intensity of defoliation in terms of the proportion of leaf area
removed rather than to a clipping height.
The objectives of the present study were to evaluate
the impacts of defoliation frequency and intensity, characterized in terms of proportion of leaf area removal, on
N uptake, N mobilization from remaining tissues, and
N allocation to growing organs. The N supply was also
considered since it alters the impact of defoliation on
N economy of the plant. The working hypothesis was that
the negative effect of defoliation on N uptake is associated
with a positive effect on N mobilization.
Plants of L. perenne, a commonly sown grass in temperate ecosystems, were grown under two contrasted N
regimes. The plants were either subject to a single defoliation, which removed a range (from 0–100%) of lamina
area, or they received four weekly defoliations at a height
of 3 cm followed by a fifth defoliation which gave a varying
range of lamina area removal. The role of the repeated
initial clipping to a set height was to create plants in which
the shoot had become morphologically adapted to defoliation. The use of 15N allowed discrimination between
the N acquired before and after defoliation and its allocation
to be traced. Following the methodological principles
proposed by De Visser et al. (1997), sampling of growing
leaves included the base inside the sheath of older leaves,
to ensure that determination of 15N mobilization would
not reflect the physical movement of meristematic leaf
tissues during their expansion from the inside to the outside of the sheaths. An additional defoliation treatment
removed 100% of the lamina plus a part of the sheath,
allowing the specific role of the sheath to be determined. In
addition to their role in supplying N to growing organs, it
was suggested that the sheath may also have a protective
role on growth of the subtending leaves (Grant et al., 1981;
Casey et al., 1999).
Materials and methods
Growth of plant material
Seeds of Lolium perenne L. were germinated on filter paper
moistened with deionized water within closed Petri dishes placed
in a growth room. The dishes were kept at 24 8C in the dark for 3 d,
then three seedlings per pot were planted in 12 cm square pots (2.9 l
capacity) containing fine sand (1.05–1.27 mm diameter). The plants
were then grown in a controlled environment growth room at
a constant 17 8C and 80% relative humidity with a 14 h photoperiod
of 500 lmol m2 s1 PAR (Photosynthetically Active Radiation)
at plant height provided by a balanced mixture of metal halide and
high pressure sodium bulbs.
Plants were watered twice each day with 40 cm3 of a complete
nutrient solution containing 0.40 mol m3 KH2PO4, 0.15 mol m3
K2HPO4, 1.0 mol m3 K2SO4, 3.0 mol m3 CaCl2, 0.50 mol m3
MgS04, 0.10 mol m3 NaCl, 0.025 mol m3 H3BO3, 0.503103 mol
m3 CuSO4, 0.002 mol m3 MnSO4, 0.503103 mol m3
Na2MoO4, 0.002 mol m3 ZnSO4, and 0.02 mol m3 FeSO4. In
addition, nitrogen was supplied as either 2 (High N) or 0.2 (Low N)
mol m3 NH4NO3. After 26 d of growth the final length of the first
and second leaf of the main tiller and the presence or absence of a
coleoptile tiller was recorded. In order to reduce variability in tiller
development within the experimental population, one plant in each
pot without a coleoptile tiller and whose leaf lengths were close
to the population mean was chosen to be retained. The two other
plants in each pot were discarded. Pots were arranged in five replicate blocks in the growth room, with one replicate of each treatment (N level3defoliation intensity3defoliation frequency, see
below) for each subsequent harvest in each block.
Defoliation treatments and 15N labelling
The defoliation treatments comprised of two parts (i) a defoliation
frequency treatment where plants were either left intact or regularly
defoliated to a set height followed in time by (ii) a defoliation intensity treatment where different percentages of leaf area were removed. As plants left intact in the defoliation frequency treatment
were subsequently defoliated on one occasion (in the defoliation
intensity treatment), they were designated as ‘single defoliation (SD)’
plants. Plants defoliated to a set height in the frequency treatment
were designated as ‘repeat defoliation (RD)’ plants.
After 45 d of growth, both Low N and High N plants were
randomly allocated to the SD and RD treatments. The SD plants were
then subject to differing intensity of defoliation where either 0%
(treatment A), 25% (treatment B), 50% (treatment C), 75% (treatment
D), or 100% (treatment E) of the lamina area was removed. This
was achieved by removing the appropriate lamina area from each
leaf. One additional intensity treatment, a cut to a height of 3 cm
above the roots (treatment F), was applied. This treatment also resulted in 100% lamina length removal, but additionally, removed a
top section of sheath material. All removed clippings were saved.
Five replicates of the SD plants subject to each intensity treatment
(A–F) were immediately harvested (day 0 after defoliation). In five
additional replicates the nutrient solution was washed from the
Defoliation impact on N dynamics in ryegrass 999
pots with three changes of deionized water. The plants were then
supplied with a nutrient solution identical to that previously supplied
except that the all nitrogen was labelled with 15N enriched to 5.5
atom%. The youngest visible leaf on each tiller was marked using
a loop of thin plastic coated wire. The plants were allowed to grow
for a further 7 d then harvested (day 7 after defoliation).
Plants allocated to the RD treatment continued to be grown in
unlabelled nutrient solution; they were clipped to a height of 3 cm
after 45, 52, 59, and 66 d of growth. On day 73, the RD plants were
submitted to the defoliation intensity treatments A, C, E, or F
described above.
The RD plants were then treated identically to SD plants with
five replicates being harvested immediately (day 0 after defoliation)
and an additional five replicates harvested 7 d later (day 7 after
defoliation) following growth in the enriched 15N nutrient solution
described above. The defoliation treatments resulted in SD and
RD plants of different ages at harvest, however, they were intended
to give plants of similar dry weight and N content (especially for
shoot material).
Harvesting and analysis of plant material
At harvest, plants were washed from the sand, and divided into
roots and shoots. Shoots were separated into two subsets of approximately equal tiller numbers, one for the estimation of the
residual lamina area, and the other one for leaf separation and 15N
analyses. With the first subset, the remaining leaves were excised
and their total area measured using a leaf area scanner using Win
Dias, Release 1.5 software (Delta-T Devices Ltd, Cambridge, UK).
This measurement on plants harvested on day 0, combined with area
measurement of the clippings on day 0, allowed calculation of the
actual percentage of lamina area removed in all defoliation treatments.
The tillers of the second subset were separated into the youngest
visible leaf (potentially containing an invisible leaf inside its sheath),
the second youngest leaf and remaining shoot material. For plants
harvested on day 7 the youngest leaf category comprised the youngest
visible leaf identified on ‘day 0’ and any new leaf which appeared
during the 7 d labelling period. The current shoot separation is
analogous to that used by Thornton and Millard (1997) and ensured
that any physical movement of N through growth did not result in
N movement between leaf categories. This separation was based
upon the expected role (sink or source) of each plant compartment
with respect to mobilization of N following defoliation. The youngest visible leaves (referred to subsequently as ‘growing leaves’) all
invariably grew and, as a consequence, would be expected to act as
a sink for mobilized N. By contrast, the remaining shoot material
comprised adult leaves in which growth was complete and would be
expected to act as a source of remobilized N. Results derived from
15
N data showed that the second youngest leaves, whose growing
status on ‘day 0’ was uncertain, behaved as the remaining shoot
material. Consequently, the second youngest leaves and remaining
shoot categories are subsequently presented together in the results as
‘adult leaves’.
After harvest, all samples were freeze-dried, weighed, and milled.
The total N and 15N content of the milled samples was determined
using a TracerMAT continuous flow mass spectrometer (Finnigan
MAT Ltd, Hemel Hempsted, England). The 15N content of the entire
plant allowed determination of root N uptake of the plants from the
start of the labelling period, according to equations described in
Millard and Neilsen (1989). Subtraction of the labelled N content
from the plants’ total N content allowed determination of the
unlabelled plant N content, which represents the N content in the
plants at the start of the labelling period (day 0). The unlabelled N
content of plants harvested on ‘day 0’ was not statistically different
from that of plants harvested on ‘day 7’, therefore from day 0 to day 7
the plant can be considered as a closed system for unlabelled N.
The partitioning of unlabelled N observed on plants harvested on
‘day 0’ was applied to unlabelled N content of plants harvested on
‘day 7’, it was then possible to calculate d7–d0 unlabelled N content
differences for each compartment. An increase in unlabelled N
content of any compartment from day 0 to day 7 was defined as
mobilization into that compartment, whilst a loss of unlabelled N
content from day 0 to day 7 was defined as mobilization out of
the compartment.
The contribution of the roots to the N mobilization into adult leaves
was calculated according to equation 1.
Root contribution ð%Þ
ðd7 d0Þ root unlabelled N content
3 100 ð1Þ
=
ðd7 d0Þ root unlabelled N content +
ðd7 d0Þ adult leaves unlabelled N content
The allocation of root uptake of labelled N to growing leaves was
calculated according to equation 2.
Labelled N allocation ð%Þ
growing leaves labelled N content
=
3 100
whole plant labelled N content
ð2Þ
Whilst the allocation of unlabelled N to growing leaves was
calculated according to equation 3.
Unlabelled N allocation ð%Þ
ðd7 d0Þ growing leaves unlabelled N content
3 100
= d0 root unlabelled N content + d0 adult
leaves unlabelled N content
ð3Þ
Statistics
Initially, analyses of variance (ANOVA) were performed for all
variables and factors except for defoliation intensity where, in order
to balance comparisons of the defoliation frequency, only treatments A, C, and E were compared against each other. Subsequently,
ANOVA were performed directly comparing treatments E and F
only. All ANOVA were performed using SAS, Release 8.01 (SAS
Institute Inc., 1999–2000).
Results
Plant dry mass, N content, and tiller number
Considered across all defoliation treatments and harvest
days, plants receiving the greater supply of N had greater
biomass and N content of both shoots and roots (P <0.01,
Tables 1, 2). However, a greater N supply not only
increased plant mass but also modified its partitioning,
increasing plant shoot/root ratio in both defoliation frequency treatments (i.e. for SD-A treatment on day 0, S/R
[HN] = 1.64 and S/R [LN] = 1.00). The effect of removing
an increasing percentage of shoot material by defoliation
on day 0 (treatments A–F) was still evident after 7 d of
regrowth, with plants subject to greater defoliation intensity
on day 0 having smaller shoot mass on day 7 (P <0.001). In
the main, defoliation frequency did not affect shoot dry
mass (P >0.05), exceptions being treatment A with the
high N supply and treatments C and E with low N on day 7,
for which shoot mass was larger under frequent than
under unfrequent defoliation. Shoot N content behaved
1000 Lestienne et al.
Table 1. The root dry mass and shoot dry mass of plants immediately after defoliation on day 0 and 7 d after the start of 15N labelling
SD: single defoliation; RD: repeated defoliation. Values are means of five replicates with SE in parentheses.
Defoliation treatment
Root dry mass (g1 plant)
High N
SD – A (0%)
SD – B (25%)
SD – C (50%)
SD – D (75%)
SD – E (100%)
SD – F (100+Sh)
RD – A (0%)
RD – C (50%)
RD – E (100%)
RD – F (100+Sh)
Shoot dry mass (g1 plant)
Low N
High N
Low N
Day 0
Day 7
Day 0
Day 7
Day 0
Day 7
Day 0
Day 7
0.60
0.67
0.54
0.62
0.58
0.62
1.13
1.00
0.98
0.98
1.90
1.53
1.46
1.04
0.87
0.82
2.23
1.67
1.27
1.23
0.16
0.17
0.21
0.17
0.22
0.17
0.43
0.36
0.38
0.27
0.35
0.31
0.26
0.25
0.20
0.22
0.54
0.51
0.37
0.33
0.99
0.92
0.67
0.56
0.38
0.32
0.98
0.67
0.39
0.45
2.83
2.31
1.98
1.53
1.28
1.00
3.25
2.14
1.34
1.07
0.16
0.14
0.13
0.08
0.07
0.05
0.20
0.12
0.09
0.07
0.33
0.27
0.22
0.20
0.14
0.14
0.34
0.26
0.18
0.16
(0.16)
(0.36)
(0.26)
(0.15)
(0.17)
(0.13)
(0.24)
(0.38)
(0.34)
(0.47)
(0.07)
(0.43)
(0.45)
(0.29)
(0.14)
(0.18)
(0.26)
(0.54)
(0.20)
(0.22)
(0.04)
(0.04)
(0.03)
(0.01)
(0.06)
(0.06)
(0.03)
(0.08)
(0.06)
(0.015)
(0.03)
(0.05)
(0.05)
(0.07)
(0.03)
(0.02)
(0.07)
(0.15)
(0.08)
(0.05)
(0.32)
(0.41)
(0.34)
(0.16)
(0.15)
(0.09)
(0.17)
(0.17)
(0.12)
(0.18)
(0.23)
(0.26)
(0.30)
(0.26)
(0.16)
(0.09)
(0.17)
(0.18)
(0.22)
(0.22)
(0.03)
(0.05)
(0.02)
(0.01)
(0.02)
(0.01)
(0.02)
(0.01)
(0.02)
(0.01)
(0.03)
(0.02)
(0.02)
(0.01)
(0.02)
(0.02)
(0.04)
(0.03)
(0.01)
(0.05)
Table 2. The amount of N in roots and shoots of plants immediately after defoliation on day 0 and 7 d after the start of 15N labelling
SD: single defoliation; RD: repeated defoliation. Values are means of five replicates with SE in parentheses.
Defoliation treatment
Root N content (mg1 plant)
High N
Day 0
SD – A (0%)
SD – B (25%)
SD – C (50%)
SD – D (75%)
SD – E (100%)
SD – F (100+Sh)
RD – A (0%)
RD – C (50%)
RD – E (100%)
RD – F (100+Sh)
13.29
13.57
12.40
13.52
11.64
12.64
18.62
18.37
17.19
15.60
Low N
Day 7
(4.23)
(3.08)
(2.96)
(1.42)
(1.59)
(0.95)
(2.73)
(3.43)
(3.86)
(3.00)
Shoot N content (mg1 plant)
24.12
21.44
20.65
17.00
13.79
15.44
25.88
21.45
19.50
18.51
(0.91)
(4.89)
(2.53)
(2.46)
(0.92)
(2.92)
(3.16)
(4.00)
(2.95)
(2.19)
High N
Day 0
Day 7
Day 0
1.63
1.61
1.97
1.81
2.06
1.70
3.39
2.98
3.23
2.82
2.93
2.95
2.48
2.49
2.15
2.26
4.50
4.60
3.20
3.01
35.34
31.60
22.22
17.73
9.92
7.97
40.23
25.08
12.40
12.30
(0.30)
(0.27)
(0.30)
(0.13)
(0.43)
(0.52)
(0.27)
(0.43)
(0.46)
(1.36)
in a similar fashion to shoot dry mass: decreasing with
defoliation intensity at both harvest dates (P <0.01 except
for single defoliation, low N on day 7, where P <0.05;
Table 2), increasing during the labelling period (P <0.001)
and mainly unaffected by defoliation frequency (P >0.05
excepted for low N treatments A and C, and high N
treatment E on day 7). For singly defoliated plants, altering
defoliation intensity caused significant differences in the
root dry mass of plants on day 7 (P <0.001; Table 1), with
the more severe defoliations resulting in the smaller root
masses. These differences still existed but were reduced in
plants previously subjected to repeated defoliation (P <0.01
for high N, P <0.05 for low N). Irrespective of N supply and
defoliation frequency, no increase in root mass over the 7
d labelling period was achieved by plants where 100% of
lamina was removed (P <0.05). Plants which were repeatedly defoliated had greater root mass on day 0 compared with plants defoliated once (P <0.05 except for high
N treatment C). Similarly, root N content was greater in
repeatedly, compared with singly, defoliated plants on day
0 (P <0.05). The root N content on day 7 was strongly
affected by the defoliation intensity received on day 0 (P
(0.27)
(0.68)
(0.51)
(0.57)
(0.38)
(0.22)
(0.34)
(0.65)
(0.64)
(0.40)
Low N
Day 7
(11.84)
(8.59)
(8.76)
(2.57)
(2.07)
(1.67)
(3.95)
(5.11)
(2.19)
(1.83)
72.06
62.90
59.97
51.85
45.39
41.77
80.26
66.87
52.57
45.73
(3.49)
(5.05)
(6.03)
(4.61)
(3.95)
(4.70)
(6.93)
(5.09)
(3.08)
(4.77)
Day 0
Day 7
3.22
2.42
2.19
1.37
0.81
0.67
4.86
2.56
1.42
1.14
6.06
5.72
4.96
5.31
4.22
3.97
6.85
6.17
4.88
4.71
(0.32)
(0.53)
(0.57)
(0.17)
(0.25)
(0.11)
(0.46)
(0.16)
(0.27)
(0.12)
(0.61)
(0.85)
(0.87)
(0.56)
(1.18)
(0.51)
(0.72)
(0.54)
(0.77)
(0.76)
<0.05; Table 2), with plants subject to the most severe
defoliations achieving no significant increase in root N
content over the 7 d labelling period.
In addition to plant mass and N content, plant tiller
number was also evaluated. Tiller number was substantially
larger for plants receiving high N than plants receiving low
N supply (P <0.001; Table 3). More remarkably, tiller
number was also much larger for plants repeatedly
defoliated than for plants defoliated once (P <0.001), despite no or only small differences in shoot mass per plant.
N uptake
Plants receiving the higher N supply took up significantly
more N during the 7 d labelling period than plants receiving
the low N solution (P <0.001; Fig. 1). Considered over both
N supplies, N uptake by singly defoliated plants was lower
than N uptake of repeatedly defoliated plants (P <0.001).
The greater amount of N taken up by repeatedly defoliated
plants was coincident with their greater initial root mass.
The response of plant N uptake to defoliation intensity was
unaltered by the previous defoliation frequency but was
Defoliation impact on N dynamics in ryegrass
Table 3. The number of tiller per plant immediately after the defoliation (day 0) and 7 d after the start of
1001
15
N labelling
SD: single defoliation; RD: repeated defoliation. Values are means of five replicates with SE in parentheses.
Treatment
Tiller number per plant
High N
SD – A (0%)
SD – B (25%)
SD – C l(50%)
SD – D (75%)
SD – E (100%)
SD – F (100%+Sh)
RD – A (0%)
RD – C (50%)
RD – E (100%)
RD – F (100%+Sh)
Low N
Day 0
Day 7
22.0
25.7
21.4
22.0
25.0
28.0
85.0
93.5
79.2
83.0
41.4
36.2
43.8
41.8
38.2
48.2
125.4
99.6
102.2
102.8
(5.0)
(4.3)
(5.8)
(5.4)
(5.0)
(5.7)
(17.4)
(18.6)
(13.8)
(23.5)
dependent on N supply. In plants receiving the high N
supply, removing all lamina area (treatments E and F) resulted in less N uptake compared with either intact plants
(treatment A) or plants where 50% of lamina area was
removed (treatment C; P <0.001). This negative effect of
defoliation intensity on N uptake in high N plants was
concomitant with the observed negative effect of defoliation intensity on root mass. By contrast, for plants receiving the low N supply, total plant N uptake was unaffected
by defoliation intensity.
Allocation of labelled N to plant compartments
Compartments of plants receiving the higher N supply
contained more labelled N than plants receiving the lower
N supply (P <0.001 for growing leaves, adult leaves and
roots). Defoliation intensity, despite affecting total N uptake of high N plants, did not affect the amount of labelled
N subsequently allocated to growing leaves (P >0.05; Fig.
2A, D). By contrast, removal of 100% of lamina area did
cause a reduction in the amounts of labelled N allocated
to roots (P <0.001; Fig. 2C, F) and to adult leaves (P <0.05
for high N and P <0.01 for low N; Fig. 2B, E). Repeatedly
defoliated plants showed a significantly greater labelled
N content in growing leaves (P <0.001 for high N, P <0.01
for low N; Fig. 2A, D) compared with plants defoliated
once. In both roots and adult leaves the previous defoliation
frequency had no effect on their labelled N content.
Mobilization of unlabelled N to new leaves
The amount of N potentially available for mobilization to
growing leaves after defoliation was the unlabelled N
content of adult leaves and roots on day 0. Increasing
either defoliation intensity or defoliation frequency did
not alter the amount of N mobilized to growing leaves
(P >0.05; Fig. 2A, D). As would be expected, as the percentage of lamina removed was increased and, in consequence, as the amount of N in remaining adult leaves was
reduced, the amount of N mobilized out of adult leaves
(9.2)
(5.7)
(17.5)
(12.3)
(5.0)
(6.1)
(9.3)
(11.7)
(18.0)
(20.3)
Day 0
Day 7
7.2
7.4
8.2
6.8
8.2
7.2
21.0
19.6
19.2
17.2
11.6
14.2
11.2
11.6
9.2
9.4
24.6
26.0
24.8
20.2
(1.1)
(0.9)
(1.3)
(0.4)
(0.8)
(1.1)
(4.6)
(4.9)
(5.7)
(4.4)
(5.2)
(3.3)
(3.9)
(2.7)
(1.6)
(3.4)
(8.0)
(8.0)
(4.1)
(2.7)
decreased (P <0.01 for high N, P >0.05 for low N; Fig.
2B, E). By contrast, plants subject to 100% of lamina area
removal (treatment E) mobilized significantly more unlabelled N from roots than plants whose leaves were left
intact (P <0.01 for HN, P <0.001 for LN; Fig. 2C, F).
Consequently, there was an increase in the relative contribution of root reserves in supplying N mobilization to
growing leaves from approximately 25% of the total up
to approximately 75% (P <0.001; Fig. 3), irrespective of
N supply (P >0.05). This increase in the contribution of
roots, both in relative and absolute terms, allowed plants
to maintain the amount of mobilized N allocated to growing leaves as defoliation intensity increased. Unlabelled N
contents on day 0 were higher in plants subject to the high
N treatment, these plants subsequently mobilized significantly more N from roots (P <0.01; Fig. 2), and adult leaves
(P <0.001; Fig. 2) to growing leaves (P <0.001; Fig. 2) than
plants receiving the lower N supply. Direct comparison of
treatment E with treatment F indicated that when 100% of
lamina area was removed, the additional removal of part
of the sheath material did not result in any significant
difference in N uptake or remobilization (P >0.05).
Relative N allocation of uptake and potential
mobilization to growing leaves
The allocation of labelled and unlabelled N to growing
leaves were calculated as a proportion of total N uptake and
the total N potentially available for mobilization, respectively. For plants receiving the lower N supply, the percentage of N taken up allocated to growing leaves increased
from approximately 60% in plants whose leaves were left
intact (treatment A) to approximately 75% in plants whose
total lamina area was removed (treatment E, P <0.001;
Fig. 4). In intact plants receiving the higher N supply,
allocation of uptake to growing leaves was greater at
70% compared with plants receiving the lower N supply
(P <0.01), this allocation also rose, though less sharply,
to 75% as the intensity of defoliation was increased
1002 Lestienne et al.
Labelled 15N uptake
(mg per plant)
50
40
30
20
10
0
A
Labelled 15N uptake
(mg per plant)
5
4
3
2
1
B
0
0
25
50
75
100
'100
+Sh'
Lamina area removed (%)
Fig. 1. Labelled 15N uptake 0–7 d after defoliation by whole plants
grown with high N supply (A) or low N supply (B) and submitted to
a single defoliation (open circles) or a regular defoliation (closed circles).
Values are means of five replicates; error bars are SE. The label ‘100+Sh’
holds for treatment ‘F’ (removal of 100% lamina area plus removal of part
of the sheath).
(P <0.001; Fig. 4). In an analogous manner, for plants
receiving the low N supply, the percentage of unlabelled N
available for N mobilization allocated to growing leaves
increased from approximately 15% in plants whose leaves
were left intact (treatment A) to approximately 35% in
plants whose total lamina area was removed (treatment E,
P <0.001; Fig. 4). The percentage allocation of potential
mobilization to growing leaves was greater in intact plants
receiving the greater N supply compared with intact plants
receiving the lower N supply (P <0.001), increasing from
30% in intact plants (treatment A) to 45% in plants where
100% of lamina area was removed (treatments E and F,
P <0.001; Fig. 4).
It is worth noting that in supplying N to growing leaves
both in intact and defoliated plants root uptake was the
major source of N compared with mobilization of N
reserves (P <0.001; Fig. 2A, D).
Discussion
Influence of defoliation intensity under high N supply
By applying defoliation intensity in terms of an increasing
percentage of leaf area removed, it allowed thresholds to be
determined above which the defoliation intensity affected
total plant N uptake. The removal of more than 75% of
the leaf area was required in order to result in a significant
reduction of N uptake in plants receiving the greater N
supply, whilst in plants receiving the lower N supply the
threshold was never reached as removal of 100% did not
reduce N uptake compared with intact plants. Many studies
have documented a negative effect of defoliation on root
N absorption by grasses when compared with intact plants
(Richards, 1993). For example, cutting Lolium perenne
plants to a height of 4 cm resulted in the cessation of
NH4NO3 uptake over the following 4 d (Ourry et al., 1988).
A single severe defoliation resulted in a large inhibition
of NO
3 uptake by L. perenne compared with undefoliated
plants for up to 8 d after defoliation (Jarvis and Macduff,
1989). Also, N uptake per plant by L. perenne or Festuca
rubra was reduced with both a single and repeat defoliation
to 4 cm (Thornton and Millard, 1997). In addition, when
evaluating the impact of defoliation severity on L. perenne,
plants cut to a 4 cm height showed a greater reduction of
N uptake compared with plants cut to a 8 cm height
(Thornton and Millard, 1996). In the present experiment,
the effect of even 100% lamina area removal was limited
compared with the literature cited above. However, this
conclusion is drawn from an integrated measurement of
N uptake over 7 d. The decrease in N uptake would probably have appeared more substantial if measured over
the first few days immediately following defoliation
(Thornton and Millard, 1997). Overall, the present results
show that the effect of defoliation on N uptake is significant only at 75% or more of leaf area removal, therefore
indicating that defoliation has a limited impact on plant
N uptake when integrated over a longer term than the
very next days following defoliation.
Defoliation of grasses has also been shown to result in
decreased root mass (Thornton and Millard, 1996) and
elongation rate (Davidson and Milthorpe, 1966; Jarvis and
Macduff, 1989; Ourry et al., 1988; Mackie-Dawson, 1999).
Indeed following defoliation of L. perenne to a 4 cm height
some root death was observed to occur over the following
13 d (Jarvis and Macduff, 1989). In the present study the
final root mass achieved on day 7 was shown to decrease as
defoliation intensity was increased across all defoliation treatments. The specific uptake of N (uptake per unit
root mass) increased with increased defoliation severity
(specific N uptake was, respectively, 17.6 and 32.8 mg
N g1 root for A and E treatments at high N, and was,
respectively, 8.1 and 13.1 mg N g1 root for A and E
treatments at low N). A positive effect of defoliation
on the specific uptake rate has previously been observed
(Macduff et al., 1989; Thornton and Millard, 1997; Pierre
et al., 2004). In part, this may have been brought about
through changes in root morphology as Dawson et al.
(2003) showed that defoliation of L. perenne resulted in
shorter and thinner roots.
N (mg per plant)
Defoliation impact on N dynamics in ryegrass
30
3
20
2
10
1
D
N (mg per plant)
A
0
0
0
0
-10
-1
B
N (mg per plant)
1003
E
-20
-2
10
1
0
0
-10
-1
C
F
-2
-20
0
25
50
75
100
'100
+Sh'
Lamina area removed (%)
0
25
50
75
100
'100
+Sh'
Lamina area removed (%)
15
Fig. 2. Labelled N content on day 7 (circles) and change in unlabelled N content between day 0 and day 7 (squares) in growing leaves (A, D), adult
leaves (B, E), and roots (C, F) of plants defoliated once only (open symbols) or regularly defoliated (closed symbols) and supplied with high N (A, B, C)
or low N (D, E, F). Values are means of five replicates; error bars are SE. The label ‘100+Sh’ holds for treatment ‘F’ (removal of 100% lamina area plus
removal of part of the sheath).
As defoliation intensity increased, the amount of root
uptake subsequently allocated to growing leaves during
the labelling period was maintained, at the expense of N
allocation to roots and to adult leaves. The decrease in N
allocation to roots was presumably related to a decreased
root sink strength, as their growth rate was reduced. Ourry
et al. (1988) demonstrated that N was mobilized from two
major compartments following defoliation: (i) stubble left
on the plant after clipping and which was preferentially
depleted, and (ii) roots. In addition to the preferential
allocation of root N uptake to new leaves, more N was
mobilized from the roots to growing leaves in response
to increasing defoliation intensity. This allowed the plants
to compensate for the increased removal of shoot N reserves as defoliation intensity increased. In the legume
Medicago sativa, a similar increased contribution of roots
in supplying mobilized N has been observed as defoliation intensity increased (Meuriot et al., 2005). The present results clearly show that defoliation intensity has
more impact on N allocation of labelled (recently taken up)
N and on N mobilization from shoots and roots than on
N uptake, allowing maintenance of N supply to growing
leaves.
In L. perenne plants subject to 100% leaf removal, the
additional removal of part of the sheath did not significantly
affect the N dynamics of the plant. One possible explanation is that the removal of the sheath per se removed
little additional mass compared with total removal of leaf
area. It also suggests that the amount of sheath removed
was insufficient to cause leaf meristem damage as this
would be expected to delay leaf growth following defoliation (Richards, 1993).
1004 Lestienne et al.
100
N allocation to growing leaves (%)
75
50
25
A
0
Root contribution (%)
100
75
50
25
0
A
75
100
50
25
B
0
0
25
50
75
100
'100
+Sh'0
Lamina area removed (%)
Fig. 3. Proportion of total unlabelled N mobilized to growing leaves
0–7 d after defoliation derived from roots only in plants grown with high
N supply (A) or low N supply (B) and submitted to a single defoliation (open circles) or a regular defoliation (closed circles). Values
are means of five replicates; error bars are SE. The label ‘100+Sh’
holds for treatment ‘F’ (removal of 100% lamina area plus removal of
part of the sheath).
Influence of plant N status
In the present experiment, the negative effect of severe
defoliation intensity on N uptake was only observed in
plants receiving the high N supply. Louahlia et al. (1999)
manipulated the N supply to two cultivars of L. perenne
such that at the time of defoliation ‘high N’ plants had
twice the N reserves of ‘low N’ plants; following defoliation all plants received an identical N supply. In both
cultivars a greater N uptake was observed in ‘low N’ plants
during the first 4 d after clipping. The results are in line with
these observations, which suggest that down-regulation of
N uptake after defoliation is either reduced or does not
occur in low N status plants; this mechanism assists defoliated plants of low N status to maintain N supply to
growing leaves.
In four grass species subject to weekly defoliation at 4
cm height it was shown that the absolute amounts of N
supplied to growing leaves after defoliation from both root
uptake and mobilization of stores were reduced in plants
receiving a reduced supply of N compared with plants
receiving a higher supply of N (Thornton et al., 1993,
1994). Uptake of N was reduced to a greater extent than
N allocation to growing leaves (%)
Root contribution (%)
100
75
50
25
0
B
0
25
50
75
100
'100
+Sh'
Lamina area removed (%)
Fig. 4. Proportion of total labelled 15N uptake (0–7 d after defoliation;
circles) and part of total unlabelled N potentially available for mobilization (on day 0; squares) allocated during the 7 d regrowth to growing
leaves of plants defoliated once only (open symbols) or regularly defoliated
(closed symbols) and supplied with high N (A) or low N (B). Values are
means of five replicates; error bars are SE. The label ‘100+Sh’ holds for
treatment ‘F’ (removal of 100% lamina area plus removal of part of the
sheath).
mobilization, as, in plants receiving the low N supply, the
proportion of the total N supplied to the growing leaves
derived from mobilization was greater. These results only
agree in part with these previous findings. They also show
that the amount of N mobilization to growing leaves was
also reduced as N supply was reduced, however, the proportion of N supplied to leaves from mobilization was
stable, irrespective of treatment, at around 38%.
Influence of defoliation frequency
In the present study, N allocation and mobilization, either
in absolute amounts or proportions, were similar for plants
subject to repeated defoliation compared with plants
Defoliation impact on N dynamics in ryegrass
clipped on one occasion, except for allocation of labelled
N to new leaves which was larger for plants repeatedly
defoliated. In addition, plants repeatedly defoliated took
up significantly more N than plants defoliated once, irrespective of N level or defoliation intensity. This difference
is most probably explained by the greater root mass of
the repeatedly defoliated plants. Mobilization out of both
adult leaves and roots was not dependant on defoliation
frequency. These results agree with Millard et al. (1990)
but not with later findings from Thornton and Millard
(1997), who found that previous frequent defoliation
resulted in post-defoliation mobilization supplying very
little or no N to the growing leaves of F. rubra and L.
perenne, respectively. Differences in experimental protocol, especially those influencing root development, may
partly explain the disparity in results. In this study, the root
mass and N contents of repeatedly defoliated plants were
greater than for single defoliated plants, whilst in Thornton
and Millard (1997) the reverse was true. The total absence
of mobilization in the frequently defoliated plants of
L. perenne in the study of Thornton and Millard (1997)
may be explained if a minimum level of N reserves exists
in roots and/or stubble below which no mobilization out
of these tissues can occur.
Despite defoliation frequency having little effect on
plant N dynamics, it highly modified plant architecture,
since tiller number was increased in plants subject to repeated defoliation compared with plants defoliated once.
Tiller number was 3.5-fold higher for plants repeatedly
defoliated than for plants defoliated once under high N
supply (respectively, 2.4-fold higher under low N supply).
By contrast, allocation of labelled N to new leaves was
increased by only 1.2-fold for frequently defoliated plants
at both N supply levels, and labelled N allocation to other
plant organs and N mobilization from adult leaves and
roots were not different between plants defoliated repeatedly or once. As a result, if considered on a tiller basis
instead of a plant basis, supply of N to growing leaves,
both from allocation of labelled N and from mobilization,
was necessarily much lower under frequent than
under single defoliation. Davies (1988) showed that an
increase in the defoliation frequency of grass swards led
to an increase in tiller density, concomitant with a decrease
in tiller mass. Tiller density and tiller mass in swards
maintained at a given herbage mass are conversely
related (Hodgson et al., 1981). A decrease in final leaf
length resultant from increased defoliation intensity and/or
frequency was observed by Van Loo (1993). The effects of decreased tiller dimensions brought about by increased defoliation frequency upon the relative use of
mobilization in supplying N to growing leaves are difficult
to predict. On the one hand decreased tiller dimensions
would probably result in less N potentially available to
be mobilized, whilst on the other hand the demand for
leaf growth would also be reduced as smaller leaves are
1005
formed. Overall, the present results show that defoliation
frequency does not substantially alter N uptake, mobilization and allocation at the plant level. However, it is remarkable that, since defoliation frequency strongly increases
tiller number per plant, N uptake, mobilization and allocation per tiller are largely altered and decreased by defoliation
frequency.
Acknowledgements
The authors are grateful to the Macaulay Development Trust and
the Région Poitou-Charentes for funding the grant to F Lestienne.
We thank the staff of the Analytical Group of the Macaulay for
technical assistance in 15N analysis.
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