Annals of Botany 88 (Special Issue): 745-751, 2001
doi:10.1006/anbo.2001.1432, available online at http://www.idealibrary.com on IE
l
®
Adaptation to Winter Stress in Nine White Clover Populations: Changes in Non-structural
Carbohydrates During Exposure to Simulated Winter Conditions and 'Spring'
Regrowth Potential
B. E. FRANKOW-LINDBERG*
Department of Ecology and Crop Production Science, Swedish University of Agricultural Sciences, Box 7043,
SE-750 07 Uppsala, Sweden
Received: 17 January 2001
Returned for revision: 22 February 2001
Accepted: 22 March 2001
Nine white clover populations: Grasslands Huia (HO, New Zealand), AberHerald (AHO, bred in the UK from
material of Swiss origin) and Sandra (SO, Sweden); sub-populations of these (survival populations) collected after
exposure to two (H2 and AH2) and four (H4, AH4 and S4) years of winter stress, respectively, at Uppsala (59°49'N,
1739'E); and a wild ecotype (WE) from the area were subjected to two simulated winter environments [+2 °C, no
light (W1) and +2/-2 C, 4 h photoperiod (W2) for 3 weeks] after hardening. After hardening, concentrations of
total non-structural carbohydrates (TNC) differed little among the populations. However, the changes in these
carbohydrates during the winter treatments differed between genetic backgrounds and populations selected for winter
survival. Populations SO, S4 and WE showed lower reductions of TNC at low constant temperatures compared with
H and AH populations. These populations were also able to maintain the TNC concentration in both stolons and
roots despite frequent exposure to sub-lethal frosts, whereas H populations and AHO and AH2 lost both starch and
water soluble carbohydrates from the stolons in treatment W2. The ability to cope with frequent sub-lethal frosts thus
appears to play a crucial role in adaptation to northern climates. There appears to have been sufficient genetic
variation in AHO for selection for this trait in the field. The amount of biomass accumulated and the rate of node
production during regrowth were generally greatest after treatment W2, when the plants tended to have the highest
concentrations of TNC.
© 2001 Annals of Botany Company
Key words: Trifolium repens L., white clover, cold tolerance, non-structural carbohydrates, starch, water soluble
carbohydrates, winter stress, spring regrowth.
INTRODUCTION
White clover (Trifolium repens L.) is a temperate species
which occurs from the Arctic to the subtropics over a wide
altitudinal range (Williams, 1987). The accumulation and
subsequent use of non-structural carbohydrates in the
stolons (Collins and Rhodes, 1995; Frankow-Lindberg
et al., 1996; Frankow-Lindberg and von Fircks, 1998;
Turner and Pollock, 1998) and in the roots (Boller and
N6sberger, 1983; Harris et al., 1983; Frankow-Lindberg,
1997; Guinchard et al., 1997) during autumn and winter
ensures the energy supply of the plant during periods of
poor radiation interception. The plant must also develop a
sufficient degree of cold hardiness to survive periods of subzero temperatures (Junttila et al., 1990). The temperature in
the field fluctuates considerably, but in a field trial
performed in Sweden (Uppsala, 59°49'N, 17°39'E) it was
rare for the temperature at the level of white clover stolons to
fall below -6
The objective of the present study was to test the hypotheses
that white clover plants adapted to long periods of winter
stress (1) have low rates of utilization of total non-structural
carbohydrate (TNC) reserves at low positive temperatures
and (2) have physiological/morphological strategies to cope
with repeated sub-lethal frosts. Changes in the concentration of TNC, divided into starch and water soluble
carbohydrates (WSC), were recorded in the stolons and
roots of three contrasting white clover cultivars, and subpopulations of these, during simulated autumn and winter
cycles in a controlled environment study. The effect of winter
environment on 'spring' regrowth potential (measured as
rate of node production and biomass accumulation) was
also evaluated. The plant material used included a cultivar
bred for the Swedish environment, cultivars bred for milder
environments, sub-populations of these that were collected
after exposure to a number of winters at Uppsala, Sweden,
and a local wild ecotype.
C, a temperature which hardened white
clover plants should be able to survive (e.g. Junttila et al.,
1990; Frankow-Lindberg and von Fircks, 1998). Despite
this, winter damage to cultivars of southern origin was
substantial in this trial (Frankow-Lindberg and von Fircks,
1998). This raises the question of how white clover plants are
affected by frequent or lengthy exposure to sub-lethal frosts.
* Fax +46 18 672209, e-mail bodil.frankow-lindberg(aevp.slu.se
0305-7364/01/100745 + 07 $35.00/00
MATERIALS AND METHODS
Plant material
The experiment included three cultivars of medium leaf size:
Grasslands Huia (H, New Zealand), AberHerald (AH, bred
in the UK from material of Swiss origin) and Sandra
(S, Sweden), sub-populations of these collected from a field
© 2001 Annals of Botany Company
746
Frankow-Lindberg--Adaptation to Winter Stress in White Clover
experiment performed in Uppsala between 1993 and 1996,
and a wild ecotype. The field experiment was sown with
Huia, AberHerald and Sandra cultivars in bi-species
mixtures together with perennial ryegrass (Lolium perenne
L. cultivar Preference) in a randomized block design, and
was harvested by mowing three four times per growing
season. The plots with species mixtures were separated by
all-grass plots and the borders of the plots were kept free
from vegetation by regular mechanical treatment. The white
clover populations were monitored by frequent samplings
during 1993 1996 and no seedlings were observed after
autumn 1993. Thus, the white clover plants in the plots can
be assumed to have established during 1993. Further details
of this experiment are given in Frankow-Lindberg and von
Fircks (1998).
Stock plants (eight genotypes per population) from
which plant material for this experiment were taken were
generated as follows: (1) Original populations were grown
from the same clover seed lots that were used to establish
the field experiment. (2) Sub-populations exposed to two
winters. Fifty stolon tips, each representing one genotype,
from plots with H and AH were collected in the field
experiment in 1995. These stolons were propagated and,
during summer 1996, each population was polycrossed
separately in an isolation house by the Legume Breeding
Group at IGER, Aberystwyth, UK. Stock plants were
grown from the seeds obtained. (3) Sub-populations
exposed to four winters. Stolon tips were again collected
in the field experiment in 1997, this time from all plots, and
these were directly propagated and grown as stock plants.
(4) In addition, eight genotypes of a wild ecotype from the
area were also collected in 1997 from a field that had been
mechanically mown during the past 15 years at similar
intervals to those used in the field experiment. These were
also propagated and grown as stock plants. Codes used for
the different populations are presented in Table 1.
All stock plants were grown in an unheated glasshouse
during the summer. Plants from populations H4, AH4 and
S4 could be expected to exhibit more clearly the traits that
were selected for in the Uppsala environment, since the
recombination of genes during polycrossing of populations
TABLE 1. Treatment codes
Treatment
Code
Huia, original population
Huia, subpopulation collected after exposure to two winters
Huia, subpopulation collected after exposure to four winters
HO
H2
H4
AberHerald, original population
AberHerald, subpopulation collected after exposure to two
winters
AberHerald, subpopulation collected after exposure to four
winters
AHO
AH2
Sandra, original population
Sandra, subpopulation collected after exposure to four winters
Wild ecotype collected from the Uppsala area
SO
S4
WE
'Winter' environment with +2 °C and no light
'Winter' environment with +2/-2 C and 4 h photoperiod
Wl
W2
AH4
H2 and AH2 might have made traits that had supposedly
been selected for in the field less discernible. A test of the
cyanogenic potential revealed great differences between the
Swedish populations on the one hand and the foreign
material on the other (Frankow-Lindberg, 1999), which
indicates that the sub-populations used were indeed true
sub-populations of the sown cultivars. There were nine
populations in total and stolon cuttings from eight separate
stock plants of each population were used for the
experiment.
Experimental setup and treatments
A total of 432 stolon tips containing one fully-expanded
leaf were planted in a fully- fertilized mixture of Perlite, peat
and sand in plastic containers (10 x 10 x 14 cm 3 ). The
pots were placed at random in an illuminated glasshouse
providing a minimum of 250 mol m -2 s PAR, and were
left to establish for 5 weeks at 18/15 C (day/night) with a
12 h photoperiod (natural photoperiod outside the glasshouse < 12 h). Plants were supplied weekly with 50 ml of a
low-N nutrient solution of the following composition:
2-3 mM N; 10 mM P; 4.9 mM K; 17 mM Mg; 58 mM Ca;
3.7 mM S; 27.0 pM Fe; 17-0 [tM Mn; 11 pM Cu; 3.2 p.M Zn:
Mo and 0-1 }IM Co during the establish22-0 tM B; 0-3 LttM
ment phase, once during hardening and once before
regrowth. Water was supplied as needed. The plants were
inoculated with a slurry containing Rhizobium (Biomethod
Research AB, Uppsala, Sweden) a week after planting.
There were no signs of mineral nutrient deficiencies and all
plants developed healthy nodules on the roots.
The hardening treatment started after 5 weeks; the
temperature in the glasshouse was reduced to + 10 C for
1 week. Plants were then moved to growth rooms with a
temperature of +2 C and an irradiance of 110 pmol m- 2
s- 1 PAR. The photoperiod was 10 h for 2 weeks and 8 h for
1 week. At the end of the hardening period (a total of 4
weeks), all fully-expanded leaves were removed and one full
set of plants (=72 plants) was subjected to a single
controlled frost of -5 C. The temperature was decreased
over a 6 h period from +2 C to the minimum temperature
(-5 C), and then increased to +2 C during the following
6 h. These plants were then placed in a heated glasshouse
and scored for survival after 2 and 4 weeks, after which they
were discarded. The purpose of this treatment was to
establish whether or not the plants had become hardened.
No damage was observed to any of the plants after a single
exposure to -5 'C.
The remaining plants were divided between two different
'winter' environment treatments. These were: (1) +2 "C and
no light (Wl); and (2) +2 CC for 12 h, and then -2 'C
every night (W2). The temperature was lowered over 4 h,
held constant for 4 h, and increased over 4 h. The
photoperiod in W2 was limited to 4 h with an irradiance
of 110 pmol m - 2 s- ' PAR; the light period occurred in the
middle of the +2 C period. 'Winter' treatments lasted for 3
weeks. All fully-expanded leaves were again removed at the
end of the 'winter' treatments. This served as a starting
point for node production but also distinguished between
old stolon material and stolon regrowth. Thereafter, plants
Frankow-Lindberg-Adaptationto Winter Stress in White Clover
747
were placed in a heated glasshouse for a 3 week regrowth TABLE 2. Starch concentration of stolons (mg g-' d. wt)
period. This coincided with spring and no supplementary from nine artificially hardened white clover populations
light was provided. All treatments are presented in Table 1.
exposed to two simulated 'winter' conditions
Measurements and analyses
Harvesting was carried out on three occasions: (1) after
hardening; (2) after 'winter' treatments; and (3) after 'spring'
regrowth (SW1 and SW2). The plants were washed free of all
soil, all remaining leaves and petioles were discarded at the
two first harvests and all stolons and roots from these
harvests were dried at 105 C for I h and thereafter at 40 °C
for 6 h. After drying, the samples were ground in a mill
through a 084 mm mesh sieve. The milled samples were
analysed for TNC (all carbohydrates except cellulose) using
the anthrone-reduction method after extraction with a weak
acid solution as described by Smith (1973), and the WSC
concentration, i.e. chiefly glucose, fructose and sucrose, was
determined using an automated anthrone procedure (Thomas, 1977). Starch levels were estimated by the difference
between TNC and WSC. The choice of preparative and
analytical methods was determined by the wish to compare
the results with data obtained in a field experiment using the
same cultivars (Frankow-Lindberg and von Fircks, 1998).
The carbohydrate analysis used does not detect pinitol;
however this occurs at a rather low concentration in white
clover (Turner and Pollock, 1998).
At the final harvest, the plants were dissected into roots,
old stolon material, stolon regrowth and leaves. The number
of apices and the number of nodes produced during
regrowth were recorded. All component parts were dried
at 105 C for 24 h and weighed separately. The rate of node
production during 'spring' regrowth (nodes per apex) was
calculated.
Data were analysed statistically by a two-way ANOVA,
using SAS (1988), as a completely randomized design with
eight replicates. Main factors were population, harvest
occasion and their interaction.
RESULTS
Starch concentration of the stolons
There were rather small differences among the populations
in the starch concentration of the stolons after the initial
hardening treatment (Table 2). However, the effects of the
treatments on starch concentration interacted strongly with
population (P < 0-001). Treatment WI generally caused a
reduction in starch concentration of the stolons (P < 005),
while treatment W2 significantly reduced starch concentrations in all H populations, AHO, AH2 and WE, but not
in AH4, SO or S4 populations (P < 0.05).
Starch concentration of the roots
There was a strong interaction between treatment and
population on root concentration of starch (P < 0.001).
After the hardening treatment, the differences among
populations were rather small (Table 3). Treatment WI
resulted in a general decrease in root starch concentration in
Population
HO
H2
H4
AHO
AH2
AH4
SO
S4
WE
Mean
Hardened
W1
W2
Mean
178
171
158
172
181
153
161
156
163
166
122
121
136
153
132
118
144
145
144
135
92
92
88
108
128
146
183
153
101*
121
131
128
127
144
147
139
163
151
136
Plants were destructively harvested after hardening and after the
respective 'winter' treatments.
Error d.f = 188, n = 8 or 7*.
LSDo.os0 = 9 (when n = 8 for both values to be compared) and = 10
(when n = 7 for one of the values to be compared).
TABLE 3. Starch concentration of the roots (mg g-' d. wt)
from nine artificially hardened white clover populations
exposed to two simulated 'winter' conditions
Population
HO
H2
H4
AHO
AH2
AH4
SO
S4
WE
Mean
Hardened
WI
W2
Mean
121*
131
146*
144
149*
144*
148
107*
109*
133
62
77
56
111
101
106
110
106
96
92
121*
126*
125
119
138
129
144
138
133
130
101
112
109
125
129
126
134
117
113
Plants were destructively harvested after hardening and after the
respective 'winter' treatments.
Error d.f= 179, n = 8 or 7*.
LSD.05 = 8 (when n = 8 for both values to be compared) and = 9
(when n = 7 for one of the values to be compared).
all populations, particularly in the H populations
(P < 005). With the exception of populations S4 and
WE, treatment W2 also reduced root starch concentration
(P < 005) in all populations. The increase in root starch
concentration in S4 and WE was significant (P < 005).
Water soluble carbohydrate concentration of the stolons
There was a strong tendency for interaction between
population and treatment on the concentration of WSC in
the stolons (P < 0.08). Thus, the WSC concentration was
generally reduced in all Grasslands Huia and AberHerald
populations after both 'winter' treatments, while only treatment W1 reduced the concentrations in the SO, S4 and WE
populations (P < 005, Table 4). However, H2 and H4 on
the one hand and AH2 and AH4 on the other contained
748
Frankow-Lindberg-Adaptationto Winter Stress in White Clover
TABLE 4. Water soluble carbohydrate concentration of
the stolons (mg g-l d. wt) from nine artificially hardened
white clover populations exposed to two simulated 'winter'
conditions
TABLE 5. Water soluble carbohydrate concentration of'
the roots (mg g-/ d. wt) from nine artificially hardened
white clover populations exposed to two simulated 'winter'
conditions
-^-^---^----
- -
-
Population
Hardened
WI
W2
Mean
HO
H2
H4
246
236
211
142
142
126
170
193
184
186
190
174
AH
AH2
AH4
230
232
245
157
194
182
181
210
208
S
S4
WE
264
245
232
197
200
176
229
246
257
Mean
238
168
209
Population
Hardened
WI
W2
Mean
HO
H2
H4
146*
142
146*
88
101
110
133*
152*
159
122
132
138
190
212
211
AHO
AH2
AH4
152
166*
133*
138
147
147
140
147
152
143
153
144
230
231
222
SO
S4
WE
160
177*
170*
153
147
138
169
176
151
161
168
153
Mean
155
130
153
Plants were destructively harvested after hardening and after the
respective 'winter' treatments.
Error d.f = 189, n = 8.
LSD. 05 = 14 (for comparing any two values).
Plants were destructively harvested after hardening and after the
respective 'winter' treatments.
Error d.f = 179, n = 8 or 7*.
LSD.o 05 = 9 (when n = 8 for both values to be compared) and = II
(when n = 7 for one of the values to be compared).
TABLE 6. Weight (g d. wt per plant) of nine artificially
hardened white clover populations exposed to two simulated
'winter' conditions
TABLE 7. The effect of hardening and winter treatments on
weight (g d. wt per plant) of white clover plants
Population
Old stolon weight
Root weight
'Spring' regrowth
Treatment
Old stolon weight
Root weight
HO
H2
H4
1.03
1-00
0-97
0.96
0.86
0.77
4.29
4-00
3.59
Hardened
1.37
0.86
W1
W2
1.17
1.24
0.73
0.69
-
AHO
AH2
AH4
1.41
1.50
1.35
0.73
0.73
0-71
4.02
4.47
3-81
SWI
SW2
1.09
1.09
0-88
0.90
3.72
4.11
SO
S4
WE
1.17
0.96
1-32
088
0-78
0-90
3-99
3-32
3.77
LSDo.os
0.04
0.03
0.10
LSDo.os0
0-06
0.04
0.21,/022*
Old stolon weight and root weight are means of three harvest
occasions (after hardening, after 'winter' treatments and after 'spring'
regrowth). 'Spring' regrowth consists of leaves + stolons produced
during the regrowth period.
Error d.f. = 307 for old stolon and root weights, and 118 for 'spring'
regrowth.
*0.22 for comparisons where HO, H2 or H4 are involved.
higher WSC concentrations after treatment W2 compared
with HO and AHO populations, respectively (P < 0.05).
Water soluble carbohydrate concentration of the roots
There was a significant interaction between treatment and
population on root concentration of WSC (P < 005). The
WSC concentration of the roots differed little among
populations after the hardening treatment, although all
Grasslands Huia populations contained slightly less
(P < 0-05, Table 5). Treatment Wl generally reduced the
WSC concentration, but less so in the H2 and H4
populations compared with HO, and AH2 and AH4
'Spring' regrowth
Means for nine white clover populations. 'Spring' regrowth consists
of leaves + stolons produced during the regrowth period. SWI and
SW2 denotes plant biomass after treatments WI and W2, respectively.
Errors d.f. = 307 for old stolon and root weights, and 118 for
'spring' regrowth.
compared with AHO (P < 0-05). Treatment W2 affected
WSC concentration less than treatment WI, and the
concentrations were higher in populations H2, H4 and
AH4 compared with HO and AHO, respectively (P < 0.05).
Old stolon weight
There was no interaction between treatment and population on old stolon weight. AberHerald populations
generally had the greatest, and Grassland Huia populations
the smallest stolon masses (P < 005, Table 6). Both winter
treatments resulted in a similar reduction in the stolon mass
of all plants (P < 005, a mean loss of approx. 12 % of the
dry weight present after hardening, Table 7). After 'spring'
regrowth, the mass of old stolon of all plants was further
reduced (P < 005, a mean loss of approx. 20 % of the dry
weight present after hardening, Table 7).
Frankow-Lindberg-Adaptation to Winter Stress in White Clover
Plant root weight
There was no interaction between treatment and population on plant root weight. It was generally smallest in all
AberHerald populations, while differences between Grasslands Huia and Swedish populations were small (P < 0.05,
Table 6). Both winter treatments resulted in a general
reduction in plant root weight (P < 005, mean of approx.
17 % of the dry weight present after hardening, Table 7),
while root weight was generally restored to pre-winter
treatment amounts after the 'spring' regrowth period
(P < 005, Table 7).
'Spring' regrowth
There was no interaction between treatment and population on 'spring' regrowth. The amount produced was
generally smallest in the survival populations H4, AH4
and S4 compared with the other populations (P < 005,
Table 6). The amount of regrowth was greater after
treatment W2 compared with treatment Wl (P < 0.01,
Table 7).
Node production rate during 'spring' regrowth
Plants from treatment W2 regrew at a faster rate than
plants from Wl (5-0 vs. 44 nodes per apex, P < 0001);
there were no interactions between treatment and population and no significant effects of population.
DISCUSSION
Both stolons and roots serve as storage organs for surplus
non-structural carbohydrate accumulation when the
demand of the plant is lower than the rate of synthesis of
these compounds (Boller and N6sberger, 1983). It is
important to bear in mind that the non-structural
carbohydrates in white clover storage organs undergo a
complex pattern of breakdown and synthesis during winter
and that the metabolism also depends on node position
within the plant (Hay et al., 1989; Turner and Pollock,
1998). This implies that any changes in the concentration of
TNC from the harvest after hardening to the harvests after
the winter treatments were the net effect of: carbon (C)
input by photosynthesis, and the utilization of C for
maintenance respiration, growth and, possibly in treatment
W2, processes related to the development of cold tolerance
(Smith, 1964). Active growth of white clover requires soil
temperatures above 5 C (Hart, 1987). Thus, the temperatures chosen in this experiment should not have induced
any substantial growth (although leaves continued to
expand) during either of the simulated winters, and any
changes in TNC concentrations recorded in treatment W2
should be primarily due to the net effect of C input by
photosynthesis less C losses due to maintenance respiration
and the possible development of cold tolerance. Net
changes in TNC between hardening and the extraction
after treatment W1 (no photosynthesis and no frosts)
should thus reflect the basal metabolism of the plants at
749
near freezing temperatures and are used here as a measure
of the maintenance respiration of the plants.
TNC after hardening
After the hardening treatment, the starch and WSC
concentrations in the stolons of all plants were high and of
a similar magnitude to those observed during autumn in the
field experiment from which the plant material used in this
experiment was derived (Frankow-Lindberg and von
Fircks, 1998). All populations had similar amounts of
TNC in both stolons and roots. This suggests that there is
little genetic variation in the capacity to accumulate reserve
carbohydrates in either stolons or roots within white clover
cultivars adapted to a broad range of environments.
After the hardening treatment, the plants exhibited a
level of cold tolerance similar to that observed during late
autumn in field-grown plants of the cultivars used
[unhardened plants of these cultivars survive to approx.
-1 C (Frankow-Lindberg and von Fircks, 1998)] and no
visible damage to any of the plants was observed after the
brief exposure to -5 C. Thus, the repeated exposure to
-2 C in treatment W2 was indeed sub-lethal and should
not have caused any frost damage to the tissues of the
plants in this experiment.
'Winter' at a low constant temperature
Starch and WSC concentrations both in stolons and
roots of all plants declined during treatment W1. The
relative reduction was greatest for starch stored in the roots,
irrespective of population. However, the effect was small in
the Swedish populations, and all Grasslands Huia populations generally lost considerably more starch and WSC
than did the AberHerald populations. Harris et al. (1983)
found that a cultivar (Olwen) originating from southern
France had a potential for greater respiration rates at low
temperatures than the cultivar S 184 which is a winter-hardy
cultivar of British origin. This suggests that a low
maintenance respiration at low positive temperatures has
an adaptive value for the survival of white clover plants at
northern latitudes. However, this trait was not selected for
in survival populations of H and AH populations.
'Winter' with repeated sub-lethalfrosts and a short
photoperiod
The Swedish cultivar was able to maintain starch and
WSC concentrations in both stolons and roots during
treatment W2. This implies that current photosynthesis was
sufficient to restore C consumed in maintenance respiration
and for the development of cold tolerance. However, all H
populations, AHO and AH2 had reduced stolon starch
concentrations in treatment W2 compared to levels following hardening, with H populations having the greatest
reductions. Population AH4, on the other hand, was able to
maintain its starch concentration in the stolon at a similar
level to that of the Swedish cultivar, which could indicate a
selection in the AH population for this characteristic. The
concentration of WSC in the stolons of all plants, on the
750
Frankow-Lindberg--Adaptationto Winter Stress in White Clover
other hand, was greater after treatment W2 than after
treatment W1. This suggests that stolon starch was broken
down into WSC at sub-freezing temperatures. Similar
results have been reported by Guinchard et al. (1997).
The ability to maintain a positive C balance in the
presence of frequent sub-lethal frosts thus appears to have a
strong adaptive value in northern climates. Higher rates of
net photosynthesis have been recorded in Sandra compared
with Grasslands Huia and AberHerald populations in
another experiment at low temperatures (Frankow-Lindberg, 1997), but when they were grown at higher
temperatures no such differences were observed (FrankowLindberg, 1999). It is hypothesized that adaptation involves
the ability to maintain net photosynthesis despite frost
events.
Biomass changes of old stolons and roots
All plants lost stolon weight during the winter treatments
and a further loss in the weight of old stolon material was
recorded during the 'spring' regrowth. Plant root weight was
also reduced during the winter treatments, but it was
restored to pre-winter treatment levels at the end of the
regrowth period. An interesting, but not quantified,
observation was that pink, healthy nodules were present
on all roots at the harvests made at the end of the hardening
treatment and also at the end of the 'spring' regrowth.
However, at the harvests made after the respective winter
treatments, there were still nodules on the roots, but these
were small and colourless. Furthermore, Gordon et al.
(1986) reported that defoliation per se induced a reduction in
the leghaemoglobin concentration of white clover nodules
and the plants used in the experiment reported here were
defoliated before winter treatments began. Thus, part of the
change in root weight was probably due to changes in the
leghaemoglobin content, a conclusion which is consistent
with the observation that the amount of a protein identified
as leghaemoglobin increased in the roots of white clover
plants during regrowth at increasing temperatures (Corbel
et al., 1999). The concentrations of TNC in the plant roots in
this study were much higher than those reported for plants
grown at higher temperatures (Boller and N6sberger, 1983;
Baur-H6ch et al., 1990). This suggests that TNC storage in
the roots complements storage in the stolons, particularly
during autumn and winter, and that the C-metabolism of
the roots during winter is important since starch stored in the
roots was, relatively, reduced more than starch stored in
the stolons in treatment W1.
'Spring' regrowth
A slightly smaller amount of 'spring' regrowth was
observed in the wild ecotype and in the populations that
were collected after exposure to four winters compared with
the original populations. Frankow-Lindberg (1999)
reported a comparatively smaller production potential in
these populations due to their short internodes. The rate of
node production during regrowth did not differ between
populations studied here. Plants from treatment W1 regrew
more slowly than plants from treatment W2, irrespective of
population. This resulted in a smaller accumulation of
biomass (on average 9 % less dry weight) in plants from
treatment W1 compared with plants from treatment W2
during the regrowth period. Both carbon and nitrogen
reserves are assumed to be mobilized during regrowth of
white clover (Volenec et al., 1996). Since N compounds
were not analysed in this experiment nothing can be said
about their role in the regrowth of the plants studied here.
However, it is pertinent that the overall concentrations of
TNC in the stolons and roots of all plants were higher after
treatment W2 than after treatment W1. In field experiments
over several seasons, the amount of spring regrowth
harvested correlated well with the concentration of TNC
in the stolons in early spring (Frankow-Lindberg et al.,
1996; Frankow-Lindberg and von Fircks, 1998). However,
it is important to note that white clover plants in the
Uppsala environment enter spring with a very small leaf
area (Frankow-Lindberg, unpubl. res.). The physiological
implications of regrowth in this situation should be similar
to that after a severe defoliation. It is well-established that
regrowth of white clover plants after defoliation mobilizes
non-structural carbohydrate reserves from both stolons and
roots, with stolon reserves being used preferentially
(Danckwerts and Gordon, 1989; Baur-Hoch et al., 1990;
Gallagher et al., 1997). The fact that the weight of old
stolons of all plants was reduced during the 'spring'
regrowth in this study suggests that compounds stored in
these played an important role in sustaining 'spring'
regrowth of the plants. In environments where the plant is
able to maintain a greater amount of its leaf area during
winter, TNC concentration may be less important in
determining the plant's rate of spring regrowth and
differences between cultivars in their ability to maintain
leaf area during winter might play a more crucial role.
In conclusion, my results support the two hypotheses
presented earlier. Thus, white clover plants adapted to an
environment with long overwintering periods have slower
rates of C reserve utilization at low positive temperatures
compared with plants of a more southern origin. However,
no selection for this characteristic among plants from
Grasslands Huia and AberHerald populations could be
detected. The ability to cope with frequent sub-lethal frosts
also appears to play a crucial role in adaptation to northern
climates. The Swedish material was able to maintain TNC
concentration in both stolons and roots when exposed to
frequent sub-lethal frosts, while the opposite was true for
non-adapted material. A selection for this characteristic was
found within the AberHerald population. The mechanism
behind this characteristic was not identified by the present
study, but it is probable that an ability to continue net
photosynthesis despite frost events is involved. This study
suggests that a greater focus on the physiology of white
clover plants when exposed to sub-lethal temperatures
would improve understanding of overwintering mechanisms. It would also be rewarding to investigate how the
absolute degree of cold hardiness is related to the response
to sub-lethal temperatures (Gusta et al., 1997). Furthermore, the relative roles of stored C and N compounds in
spring regrowth in different environments remain to be
resolved (Bouchard et al., 1998).
Frankow-Lindberg-Adaptationto Winter Stress in White Clover
ACKNOWLEDGEMENTS
I thank the Behms Foundation for financial support, the
Institute of Grassland and Environmental Research in
Aberystwyth for performing the carbohydrate analyses, the
Legume Breeding Group at the same institute for developing and supplying seed of H2 and AH2, A. Andersson, Y.
Schang, S. Eriksson and A. Hansson for skilful technical
assistance, G. Ekbom and B. Vegerfors for statistical advice
and Dr. A. Lfischer for helpful criticism.
LITERATURE CITED
Baur-Hoch B, Michler F, N6sberger J. 1990. Effect of carbohydrate
demand on the remobilization of starch in stolons and roots of
white clover (Trifolium repens L.) after defoliation. Journal of
Experimental Botany 41: 573-578.
Boiler BC, Niisberger J. 1983. Effects of temperature and photoperiod
on stolon characteristics, dry matter partitioning, and nonstructural carbohydrate concentrations of two white clover
ecotypes. Crop Science 23: 1057-1062.
Bouchard V, Macduff JH, Ourry A, Svenning MM, Gay J, Simon JC,
Boucaud J. 1998. Seasonal pattern of accumulation and effects of
low temperatures on storage compounds in TriJblium repens.
Physiologia Plantarum 104: 65-74.
Collins RP, Rhodes I. 1995. Stolon characteristics related to winter
survival in white clover. Journal of Agricultural Science 124:
53-62.
Corbel G, Robin Ch, Frankow-Lindberg BE, Ourry A, Guckert A. 1999.
Regrowth of white clover after chilling: assimilate partitioning and
vegetative storage proteins. Crop Science 39: 1756-1761.
Danckwerts JE, Gordon AJ. 1989. Long-term partitioning, storage and
remobilization of 14C assimilated by Trifolium repens (cv. Blanca).
Annals of Botany 64: 533-544.
Frankow-Lindberg BE. 1997. Assimilate partitioning in three white
clover cultivars in the autumn, and the effect of defoliation. Annals
of Botany 79: 83 87.
Frankow-Lindberg BE. 1999. Effects of adaptation to winter stress on
biomass production, growth and morphology of three contrasting
white clover cultivars. Physiologia Plantarum 106: 196 202.
Frankow-Lindberg BE, Salomonsson L, Torstensson A. 1996. The
relationship between stolon characteristics, total non-structural
carbohydrates, winter survival and spring yield of white clover in
Sweden. In: Frame J, ed. Recent research and development on white
clover in Europe. REU Technical Series 42: 51-54.
751
Frankow-Lindberg BE, von Fircks HA. 1998. Population fluctuations in
three contrasting white clover cultivars under cutting, with
particular reference to overwintering properties. Journal of
AgriculturalScience 131: 143-153.
Gallagher JA, Volenec JJ, Turner LB, Pollock CJ. 1997. Starch
hydrolytic enzyme activities following defoliation of white clover.
Crop Science 37: 1812-1818.
Gordon AJ, Ryle GJA, Mitchell DF, Lowry KH, Powell CE. 1986. The
effect of defoliation on carbohydrate, protein and leghaemoglobin
concentration of white clover nodules. Annals of Botany 58:
141-154.
Guinchard MP, Robin Ch, Grieu Ph, Guckert A. 1997. Cold acclimation
in white clover subjected to chilling and frost: changes in water
and carbohydrate status. European Journal of Agronomy 6:
225-233.
Gusta LV, O'Connor BJ, MacHutcheon MG. 1997. The selection of
superior winter-hardy genotypes using a prolonged freezing test.
Canadian Journal of Plant Science 77: 15-21.
Harris W, Rhodes I, Mee SS. 1983. Observations on environmental and
genotypic influences on the overwintering of white clover. Journal
of Applied Ecology 20: 609-624.
Hart AL. 1987. Physiology. In: Baker MJ, Williams WM, eds. White
clover. Wallingford: CAB International, 125-151.
Hay MJM, Chu ACP, Knighton MV, Wewala S. 1989. Variation with
season and node position in carbohydrate concentration of white
clover stolons. XVI InternationalGrasslandCongress, 4-11 October
1989, Nice, France. Association Frangaise pour la Production
Fourragere, 1059-1060.
Junttila O, Svenning MM, Solheim B. 1990. Effects of temperature and
photoperiod on frost resistance of white clover (Trifolium repens)
ecotypes. Physiologia Plantarum79: 435-438.
SAS. 1988. SAS's user's guide. Statistics. Cary, NC: SAS Institute.
Smith D. 1964. Winter injury and the survival of forage plants.
Herbage Abstracts 34: 203-209.
Smith D. 1973. The nonstructural carbohydrates. In: Butler GW,
Bailey RW, eds. Chemistry and biochemistry of herbage. Vol. 1.
London: Academic Press, 106-155.
Thomas TA. 1977. An automated procedure for the determination of
soluble carbohydrates in herbage. Journal of the Science of Food
and Agriculture 28: 639 642.
Turner LB, Pollock CJ. 1998. Changes in stolon carbohydrates during
the winter in four varieties of white clover (Trifolium repens L.)
with contrasting hardiness. Annals of Botany 81: 97-107.
Volenec T, Ourry A, Joern BC. 1996. A role for nitrogen reserves in
forage regrowth and stress tolerance. Physiologia Plantarum 97:
185 193.
Williams WM. 1987. Adaptive variation. In: Baker MJ, Williams WM,
eds. White clover. Wallingford: CAB International, 299-321.