Annals of Botany 92: 537±545, 2003
doi:10.1093/aob/mcg165, available online at www.aob.oupjournals.org
The Role of Roots and Cotyledons as Storage Organs in Early Stages of
Establishment in Quercus crispula: a Quantitative Analysis of the Nonstructural
Carbohydrate in Cotyledons and Roots
D A I S U K E K A B E Y A 1 , * and S A T O K I S A K A I 2
1Department of Ecology and Evolutionary Biology, Graduate School of Science, Tohoku University, Aoba,
Sendai 980-8578, Japan, 2Graduate School of Life Science, Tohoku University, Aoba, Sendai 980-8578, Japan
Received: 20 January 2003 Returned for revision: 23 April 2003 Accepted: 10 June 2003 Published electronically: 7 August 2003
Quercus seedlings have hypogeal cotyledons and tap roots, both of which act as storage organs. The importance
of the storage function in the two organs may change as the seedling develops. Therefore, changes in carbohydrate reserves in cotyledons and roots of Q. crispula grown under 40 % and 3 % of full light from shoot
emergence to the completion of the ®rst leaf ¯ush were monitored. In addition, a shoot-clipping treatment was
performed to examine the relative contribution of the cotyledons and tap roots to resprouting. Cotyledons maintained large amounts of nonstructural carbohydrates during shoot development, and carbohydrates were still
present in the cotyledons during the ®nal phase of leaf ¯ush. In addition, a notable increase in the amount of
carbohydrates was observed in tap roots before leaf ¯ush at both light levels. Since root development occurred
before leaf ¯ush, even in plants grown under 3 % light, the carbohydrate found in them presumably originated
from seed reserves and was translocated to roots as storage reserves. When shoots were clipped at the leaf
¯ushing stage, the amount of carbohydrate decreased only in the cotyledons after resprouting, suggesting that
cotyledons act as the main storage organs during shoot development stages. However, it could be advantageous
as a `risk avoidance strategy' for the seedlings to store reserves in both cotyledons and roots, since cotyledons
may be removed by predators during shoot development.
ã 2003 Annals of Botany Company
Key words: Quercus crispula Blume, Mizunara oak, seedling, carbohydrate storage, germination stage, hypogeous
plant, resprouting, shoot destruction, TNC, seed reserves.
INTRODUCTION
Quercus species have well-developed tap roots at the
seedling stage (Abrams, 1996; Larsen and Johnson, 1998),
and this feature ®rst appears in the germination-establishment stages (Jones, 1959; Crow, 1988). In addition to
helping withstand drought (Rao, 1988; Callaway, 1992;
Canham et al., 1996), storage has been regarded as an
important function of the tap roots in Quercus species
(Jones, 1959; Matsubara and Hiroki, 1985; Crow, 1988;
Larsen and Johnson, 1998). Storage functions in tap roots
have been shown in seedlings older than 1 year, and saplings
(Kruger and Reich, 1993; Sakai et al., 1997; Canham et al.,
1999), but there have been few studies concerning the
importance of the tap roots as storage organs during
germination to establishment stages.
Quercus species generally have large seeds. In several
large-seeded species, a large amount of seed biomass
remains in cotyledons even after the development of
functional shoots. Several ecologists have argued that the
biomass remaining in cotyledons provides a reserve that can
be used for growth and survival of seedlings in the presence
of stress factors, such as low light, frequent shoot destruction, and strong competition (Grime and Jeffrey, 1965;
Janzen, 1976; Brookes et al., 1980; Westoby et al., 1992;
* For correspondence: Forestry and Forest Products Research Institute,
Kiso Experiment Station, 5473-8 Kisofukushima, Kiso, Nagano 397-0001,
Japan. E-mail [email protected]
Leishman and Westoby, 1994; Kitajima, 1996; Dalling
et al., 1997; Frost and Rydin, 1997; Hoshizaki et al., 1997;
Bon®l, 1998; Dalling and Harms, 1999). However, the
importance of the storage function of cotyledons remains
controversial. To evaluate the importance of the biomass
remaining in the cotyledons several studies have been
carried out, in which cotyledons were removed when
seedlings had almost developed functional shoots. Some
of these studies have suggested that the residual cotyledon
biomass may be bene®cial for the growth and survival of
seedlings growing under stressful conditions (Bon®l, 1998;
Dalling and Harms, 1999). In addition, Bon®l (1998)
showed that, in Quercus laurina, cotyledon removal can
severely reduce the survival rates of seedlings, even under
moderate conditions. In studies of Q. robur, however, the
results are mixed; in some cases cotyledon removal has not
affected seedling growth and survival, nor regrowth after
defoliation (Sonesson, 1994; Andersson and Frost, 1996),
but in an investigation by Frost and Rydin (1997) cotyledon
removal did reduce the growth both of seedlings subjected
to defoliation and seedlings that were adversely affected by
root competition.
This study is based on the hypothesis that the importance
of storage in cotyledons varies, depending on the availability of storage substances in the tap roots. Therefore, to
evaluate the relative importance of the storage function in
cotyledons and tap roots, changes in carbohydrate storage
levels in cotyledons and roots of Quercus crispula were
Annals of Botany 92/4, ã Annals of Botany Company 2003; all rights reserved
538
Kabeya and Sakai Ð Roots and Cotyledons as Storage Organs in Quercus crispula
monitored from germination to the development of a
functional shoot. A shoot clipping experiment to examine
the relative contributions of the reserves in cotyledons and
tap roots during resprouting was also performed.
MATERIALS AND METHODS
Plant material
Quercus crispula is a common deciduous broad-leaved tree
species in the cool temperate forests of Japan. The species
shows a ¯ush-type shoot elongation pattern (Kikuzawa,
1983), and the seedlings' shoots usually elongate once in
spring on natural forest ¯oors. However, amongst seedlings
growing under favourable light conditions (e.g. in a gap
created by a tree fall), there is often a further period of shoot
elongation at some stage during the growing season.
Considerable numbers of seedlings lose their shoots through
herbivory and shoot dieback, especially during the autumn
and the following early spring (Kabeya, 2001). Like other
Quercus species, the species has hypogeous cotyledons, and
the seedlings produce at least two lateral buds around the
root collar (i.e. below ground). In addition, they have four or
®ve scaly leaves with lateral buds on their epicotyl.
Following shoot destruction, they can resprout from these
dormant buds. Thus, above-ground damage usually causes
very little meristematic limitation in this species, although
Hasegawa (1984) reported that Q. crispula seedlings could
not resprout when they were clipped below the root collar.
Plant cultivation
Experiments were conducted in the Hakkoda Botanical
Laboratory (40°38¢N, 140°51¢E, altitude 900 m) in 1999 and
2001. Acorns used in these experiments were collected from
the eastern side of lake Towada (40°26¢N, 140°56¢E,
altitude 400 m) in the autumns of 1998 and 2000 for the
1999 and 2001 experiments, respectively. Collected acorns
were washed and stored in a dark cold-room (<5 °C) until
sowing. Before sowing, the fresh mass of each acorn was
measured. The mean fresh mass of the acorns was 3´8 and
2´2 g in 1999 and 2001, respectively, and there was a
signi®cant difference between these years (ANOVA,
F = 51´9, P < 0´001). In 1999, to estimate the contribution
of the initial cotyledon dry mass to the fresh mass of the
acorn, the fresh mass of 12 acorns was measured, they were
then dried in an oven at 70 °C for 3 d, and re-weighed. The
relationship between the fresh mass of the acorn (including
the seed coat) and the cotyledon dry mass was found to be:
cotyledon dry mass (mg) = 392´5 3 acorn fresh mass (g)
± 233´4 (R2 = 0´96).
One acorn was sown per pot (12 3 14 cm) ®lled with
AKADAMA soil : sand (2 : 1, v/v). No fertilizer was
supplied during the experiment.
In 1999, approx. 500 acorns were sown. To evaluate the
effect of photosynthesis on the growth and amount of
storage resources, pots were divided into two groups, one of
which was placed in 40 % of full light, and the other in 3 %
of full light using shade cloths to set the light levels.
Seedlings were checked every day to determine the date of
shoot emergence (when the tip of the hypocotyl emerged
from the seed coat). In 2001, approx. 150 acorns were sown
on 14 June 2001. To minimize the effect of the seedlings'
photosynthesis on carbohydrate storage (see Results), all of
these seedlings were subjected to 3 % light.
Harvest of control seedlings
To evaluate changes in biomass distribution and the
levels of carbohydrate reserves in roots and cotyledons,
unclipped seedlings were harvested six times in 1999 and
twice in 2001. Hereafter, these seedlings are referred to as
control seedlings to distinguish them from the clipped
seedlings (see below).
In 1999, most of the seedlings' shoots emerged 10±22 d
after sowing. Here, to reduce the effects of ontogenical
differences, seedlings that emerged on similar dates were
collected. Since the numbers of single cohort seedlings (i.e.
seedlings that sprouted within a few days of each other)
were insuf®cient for the experiment, two seedling cohorts
were used. Cohorts 1 and 2 consisted of seedlings with
shoots that emerged between 29 June and 2 July, and 5 July
and 10 July, respectively. Seven seedlings growing under
each of the two light conditions were collected in each
harvest. The cohort 1 seedlings were collected on 7 July,
21 July and 12 Aug. (1, 3 and 6 weeks after shoot
emergence, respectively), while the cohort 2 seedlings were
harvested on 7 July, 21 July and 5 Aug. (0, 2 and 4 weeks
after shoot emergence, respectively). Seedlings were
selected randomly from each cohort.
At each harvest, seedlings were collected in the morning,
brought back to the laboratory, separated into leaves, stems,
roots, cotyledons and seed coats, and placed in an oven
within 6 h of collection. The seedlings were dried at 70 °C
for 3 d then weighed. After weighing, roots and cotyledons
of each seedling were used for carbohydrate analysis (see
below).
In 2001, shoot elongation started in almost all of the
seedlings 11±13 d after sowing (25±27 June); these
seedlings were regarded as a single cohort. Six control
seedlings from each light treatment were collected on
19 July and 8 Aug. (3 and 6 weeks after shoot emergence,
respectively). In both harvests, the same protocols were
used as in 1999.
Clipping experiment
To examine the relative contribution of the reserves in the
cotyledons and roots to resprouting, shoot clipping experiments were carried out in both 1999 and 2001.
In 1999, 20 seedlings of cohort 1 were selected randomly
from each light treatment and their epicotyls were clipped
1´5 cm above the cotyledonary node (root collar) on 22 July
(3 weeks after shoot emergence). Leaf expansion of each
seedling was approx. 50 % complete at this time. Clipped
seedlings were checked daily until 12 Aug. to observe
whether they had resprouted. The date of bud break and the
date that leaf ¯ush began were recorded. On 12 Aug.,
Kabeya and Sakai Ð Roots and Cotyledons as Storage Organs in Quercus crispula
seedlings that had resprouting shoots exceeding 0´5 cm in
length, or in which leaf ¯ush had started, were harvested.
Harvested seedlings were separated into roots, cotyledons,
stems, resprouting stems and resprouting leaves. They were
then dried and weighed. Weighed roots and cotyledons of
seven randomly selected seedlings in each light environment were used for carbohydrate analysis. The seedlings
that did not resprout by 12 Aug. were checked for
resprouting every 2 weeks until the end of the growing
season (4 Oct.).
In 2001, clipping treatments were applied on two
different dates: 19 July (the 3-week clipping) and 8 Aug.
(the 6-week clipping). Mice gnawed the shoots of some
seedlings and/or removed their cotyledons during the
experimental period. For this reason, only 14 and 15
seedlings were subjected to the 3-week clipping and 6-week
clipping treatments, respectively. On 14 Aug. (4 weeks after
the 3-week clipping) and 30 Aug. (3 weeks after the 6-week
clipping), seedlings with resprouting shoots that exceeded
0´5 cm in length or in which leaf ¯ush had started were
harvested. All seedlings that had not resprouted by the
harvest times were re-examined to see if they had begun to
resprout at the end of the growing season (27 Sept.). The
harvested seedlings were treated in the same way as those in
the 1999 experiment. In this case, however, the roots and
cotyledons of all harvested seedlings were used for
carbohydrate analysis.
Carbohydrate analysis
To determine the amount of carbohydrate reserves, the
concentrations of total nonstructural carbohydrate (TNC) in
cotyledons and roots were analysed by enzymatic hydrolysis
methods similar to those of Ono et al. (1996). Roots and
cotyledons were ground using a Wiley mill, passed through
a 65-mesh screen and dried in the oven for 24 h. Samples
(2±3 mg) were homogenized with 80 % ethanol. After the
ethanol had been removed by evaporation, they were
suspended in 0´5 ml of 0´2 M KOH and placed in boiling
water for 30 min. After cooling, 2´00 ml of 1´0 M acetic acid
was added. To digest starch to glucose, 0´5 ml of Rhizopus
amyloglucosidase (Sigma, St Louis, USA) solution (35 units
ml±1 in 50 mM Na acetate buffer, pH 4´5) was added, and the
tubes were incubated at 55 °C for 30 min. After digestion,
the tubes were placed in boiling water for 1 min and then
centrifuged. The supernatant was transferred to another
tube, and its total sugar content was determined by the
phenol±sulfuric acid method (Dubois et al., 1956).
Absorbance was measured at 490 nm in a spectrophotometer (UV-16A; Shimadzu Co., Japan), using a glucose
solution as a calibration standard. TNC pools, i.e. the actual
amounts of TNC in the cotyledons and roots, were
calculated by multiplying the concentration of TNC by the
respective dry masses of these organs.
In control seedlings harvested 3 weeks after shoot
emergence (and also 6 weeks after shoot emergence in
2001), and resprouting seedlings in both years, the starch
concentrations of cotyledons and roots were determined as
follows:
539
starch concentration (glucose equivalent) =
glucose concentration after starch hydrolysis
± glucose concentration before starch hydrolysis
where the glucose concentration before starch hydrolysis
was determined from the subsamples collected from the
supernatant of sample liquid before the addition of
amyloglucosidase solution in the method described above.
For the determination of glucose concentrations, a glucoseoxidase kit (Glucose B test; Wako Co., Osaka, Japan) was
used. Starch pools in the various organs were calculated by
multiplying the concentration of starch by the respective dry
masses.
Statistical analysis
The effects of light conditions and harvest time (i.e.
seedling development stages) were tested on plant dry
masses, TNC concentrations in cotyledons and roots, and
TNC pools in cotyledons and roots. For this, two-way
ANOVA was used, with light environment (40 % and 3 %)
and harvest time (0, 1, 2, 3, 4 and 6 weeks after shoot
emergence) as the main effects, with subsequent Games±
Howell post hoc test. In the 1999 clipping experiment, to
assess the contribution of carbohydrate storage in cotyledons and roots to resprouting, two comparisons were
performed. In the ®rst, TNC (starch) concentrations and
pools were measured in the two organs of the seedlings at
two developmental stages: the levels at the time of clipping,
and levels after resprouting (designated `initial' and
`resprouting' levels, respectively). In the second comparison, TNC concentrations and pools were compared between
treatments (i.e. between the resprouting seedlings and the
unclipped (control) seedlings, both of which were harvested
at the same time). For this, two-way ANOVA was used,
with light environment and seedling status (initial, resprouting and control) as the main effects, and each comparison
between seedlings of differing types was tested by Dunnett
post hoc tests. In the 3-week clipping experiment in 2001,
the results were also evaluated by one-way ANOVA, with
seedlings status as the main effect, to clarify the effect of
resprouting on the carbohydrate stored in the cotyledons and
roots. To analyse differences between TNC concentrations
and pools in roots and cotyledons, the Wilcoxon sign rank
test (Sokal and Rohlf, 1995) was used. To analyse
differences in resprouting shoot mass between light conditions (in 1999) and clipping periods (in 2001), the Mann±
Whitney U-test was applied. SAS software (SAS 1989) was
used for all statistical analyses except the Games±Howell
post hoc tests, which were calculated by StatView software
(SAS, 1998), and the Wilcoxon sign rank tests.
RESULTS
Control seedling development after shoot emergence
The total dry mass of the control seedlings (i.e. shoot and
root dry mass, excluding cotyledons) increased as seedling
development progressed, under both 40 and 3 % of full light
(Fig. 1A). Leaf ¯ushing started 2 weeks after shoot
540
Kabeya and Sakai Ð Roots and Cotyledons as Storage Organs in Quercus crispula
TA B L E 1. F statistics (with probabilities) for a two-way
ANOVA testing the effects of the number of days from the
start of shoot emergence to harvesting (Harvest, H), light
conditions (Light, L), and their interaction, on dry mass
(total, shoot and root), the percentage of cotyledon dry mass
in dry mass at sowing (estimated from fresh mass), TNC
concentrations in cotyledons and roots, and TNC pools in
cotyledons and roots of the seedlings in 1999
Harvest (5)*
F
P
Light (1)*
Dry mass
Total
45´8 <0´001 5´7
Shoot
40´2 <0´001 0´9
Root
31´3 <0´001 12´5
Percentage of cotyledon mass 58´7 <0´001 2´1
TNC concentrations
Cotyledon
47´7 <0´001 3´6
Root
2´7 0´029
2´9
TNC pools
Cotyledon
30´5 <0´001 2´0
Root
16´1 <0´001 12´1
0´019
0´346
0´001
0´154
H 3 L (5)*
2´3
0´3
6´7
1´6
0´051
0´928
<0´001
0´176
0´062 2´4
0´095 2´8
0´045
0´024
0´157 2´4
0´001 5´9
0´042
<0´001
* Degrees of freedom.
F I G . 1. Total, shoot and root dry mass of the Q. crispula seedlings
growing in 40 and 3 % of full light in 1999 (mean 6 s.e., n = 7) (A, B
and C, respectively). Symbols with different letters are signi®cantly
different at the P < 0´05 level according to the Games±Howell test.
* The same letters apply to both light symbols; ** results from the two
light treatments were pooled.
emergence and was complete within 4 weeks of shoot
emergence. From 0 to 3 weeks after shoot emergence, the
total dry mass of the control seedlings grown under 40 and
3 % light was similar. From 4 weeks after shoot emergence,
the total dry mass of the control seedlings growing in 40 %
light tended to be greater than that of seedlings in 3 % light,
although there was only marginal interaction between
harvest time and light (Table 1). In 3 % light, root dry
mass did not increase after 2 weeks from shoot emergence
(Fig. 1C). In addition, the root dry mass was greater in 40 %
light than in 3 % light at 4 weeks after shoot emergence, but
the shoot dry mass did not differ between the environments
during the developmental period (Fig. 1B and Table 1).
The dry mass of the cotyledons decreased as the seedlings
developed (Fig. 2), and the rate of reduction was similar in
F I G . 2. Changes in the percentage of dry mass remaining in the
cotyledons (mean 6 s.e., n = 7). Seedlings were grown in 40 % and 3 %
of full light. The initial dry mass of the cotyledons (cotyledon dry mass
at sowing) was calculated from the regression of dry mass of cotyledon
on fresh mass of the acorn. Symbols with different letters are
signi®cantly different at the P < 0´05 level according to the Games±
Howell test (both light conditions pooled).
both 40 and 3 % light (Table 1). However, 30 % of the initial
dry mass remained in cotyledons 6 weeks after shoot
emergence, by which time shoot development was already
complete (Fig. 2).
Substantial concentrations of TNC were observed in the
roots of the control seedlings throughout the experiment
period in both 40 and 3 % light (Fig. 3). We found
signi®cant effects of harvest time on the root TNC
concentrations in 40 % light (one-way ANOVA, P =
0´013). but not in 3 % light (P = 0´124); i.e. root TNC
concentrations tended to increase slightly throughout the
experimental period in 40 % light, but not in 3 % light
(Fig. 3). TNC concentrations in cotyledons decreased during
Kabeya and Sakai Ð Roots and Cotyledons as Storage Organs in Quercus crispula
541
F I G . 3. TNC concentrations in cotyledons and roots of the seedlings
growing in 40 and 3 % of full light in 1999 (mean 6 s.e., n = 7).
F I G . 5. Dry mass of the resprouted shoot (leaves + stems) of the
seedlings in shoot clipping treatments in 1999 and 2001. Means and s.e.
are shown as columns with error bars. In 1999, all seedlings were clipped
3 weeks after shoot emergence, and in 2001 seedlings growing under 3 %
of full light were clipped 3 and 6 weeks after shoot emergence.
light, and there was no signi®cant difference between the
TNC pools in the cotyledons and roots in the two light
treatments at that time (Wilcoxon sign rank test, P = 0´08
and 0´11 in 40 and 3 % light, respectively).
Clipping treatment
F I G . 4. TNC pools (log-scale) in cotyledons and roots of the seedlings
growing in 40 and 3 % of full light in 1999 (mean 6 s.e., n = 7).
the experimental period in both 40 and 3 % light (Fig. 3;
Table 1), but the relative difference between the light
environments varied at different harvest times (as shown by
the signi®cant interaction in Table 1). TNC concentrations
in cotyledons were as low as those in the roots 6 weeks after
shoot emergence (Wilcoxon sign rank test, P = 0´13 and
0´08 in 40 and 3 % light, respectively).
The TNC pools (i.e. the amounts rather than concentrations of TNC) in the roots of the control seedlings in both
40 and 3 % light increased at similar rates for the ®rst
2±3 weeks from shoot emergence (Fig. 4). However, after
4 weeks in 40 % light the TNC pools in the roots had
continued to increase, whereas those in 3 % light grew much
more slowly. The TNC pools in the cotyledons declined
throughout the developmental process in both light treatments (Fig. 4), although the relative difference between the
light environments varied at different harvest times
(Table 1). Six weeks after shoot emergence, more than
90 % of the initial TNC pool (i.e. the TNC pool at 0 weeks)
in the cotyledons had been consumed in both 40 % and 3 %
In 1999, 17 out of 20 seedlings had resprouted in 40 %
light and 14 out of 20 had resprouted in 3 % light, 3 weeks
after shoot clipping (12 Aug.). By the end of the growing
season (4 Oct. 2001), 19 seedlings had resprouted in both
40 % and 3 % light. In 2001, eight out of 14 and ®ve out of
15 seedlings from the 3-week and 6-week clipping
treatments, respectively, had resprouted. At the end of the
growing season (27 Sep. 2001), bud swellings were
observed in one and ®ve seedlings that had not resprouted
after the 3-week and 6-week clippings, respectively. In
1999, there was no signi®cant difference between the mean
dry masses of resprouting shoots in the 40 % and 3 % light
treatments (Mann±Whitney U-test; P = 0´88, Fig. 5). In
2001, the dry mass of the resprouted shoots in 3 % light that
had been clipped at 6 weeks was lower than that of the
seedlings clipped at 3 weeks (P = 0´05, Fig. 5).
In 1999, the concentration of TNC in cotyledons of the
resprouting seedlings was signi®cantly lower than initial
levels (obtained from control seedlings harvested 3 weeks
after emergence) when data from seedlings in both light
environments were pooled (Table 2; Fig. 6A). Although the
TNC concentration in the cotyledons tended to be higher in
the resprouting seedlings than the control seedlings (i.e.
those harvested 6 weeks after emergence) particularly in 3 %
light, this difference was not statistically signi®cant. The
TNC pools in the cotyledons showed the same trends as the
TNC concentrations in the cotyledons (Fig. 6B). Although
the trends shown by starch concentrations and pools in
cotyledons were the same as those of TNC concentrations
and pools in the cotyledons, the light environment and
542
Kabeya and Sakai Ð Roots and Cotyledons as Storage Organs in Quercus crispula
seedling status showed signi®cant interactions in this case
(Table 2). Furthermore, the differences between initial
starch concentration and pools in cotyledons and those
found in resprouting seedlings were statistically signi®cant
only for seedlings grown in 3 % light (Dunnett test, P <
0´05). In contrast, the concentrations and pools of TNC and
TA B L E 2. F statistics (with probabilities) for a two-way
ANOVA of the effects of seedling status (Status, S) (i.e.
initial, control or resprouting, described in Fig. 6), light
conditions (Light, L), and their interaction, on TNC
concentrations and pools in cotyledons and roots of the
seedlings in 1999
Status (2)*
TNC concentrations
Cotyledon
Root
TNC pools
Cotyledon
Root
Starch concentrations
Cotyledon
Root
Starch pools
Cotyledon
Root
F
P
10´2
1´6
<0´001
0´224
5´7
11´5
0´960
0´888
2´3
6´1
0´185
0´081
8´4
6´8
0´001
0´003
2´1
3´8
0´588
0´125
1´6
2´3
0´283
0´012
18´7
4´6
<0´001
0´017
2´2
1´8
0´054
0´530
2´7
2´4
0´015
0´003
11´9
9´2
<0´001
0´001
11´1
0´058
0´031
5´8
0´041
0´001
* Degrees of freedom.
Light (1)*
S 3 L (2)*
starch in roots were not lower than initial levels at the time
of resprouting in either 40 or 3 % light (Fig. 6C and D).
Furthermore, in 40 % light, TNC pools, starch concentrations and starch pools in roots of resprouting seedlings were
lower than those of control seedlings (P < 0´05). The results
of the 3-week clipping treatment in 2001 were similar to
those obtained in 1999 (Fig. 7), but signi®cant differences
were found only in the comparison between TNC pools in
cotyledons of resprouting seedlings and initial levels
(Dunnett test P < 0´05).
Following the 6-week clipping, TNC concentrations in
both roots and cotyledons tended to decrease after
resprouting (Fig. 7). However, the detected changes were
not statistically signi®cant (Mann±Whitney U-test, P = 0´14
and 0´18 for cotyledons and roots, respectively). Similar
trends were found in TNC pools in both roots and
cotyledons (P = 0´12 and 0´24 in cotyledons and roots,
respectively).
DISCUSSION
Importance of the cotyledons as storage organs in the early
stages of seedling development
Considerable amounts of nonstructural carbohydrate were
shown to be present in the cotyledons and roots of
Q. crispula during the ®nal phase of shoot development
(3±4 weeks after shoot emergence, Fig. 4). Of these
carbohydrates, only those in the cotyledons decreased
after resprouting (Fig. 6). This suggests that the carbohydrate reserves in the hypogeal cotyledons, rather than the
F I G . 6. TNC concentrations and pools in cotyledons and roots of the seedlings at the time of shoot clipping (initial), seedlings after resprouting
(resprouting) and control (unclipped) seedlings harvested at the same time as resprouting (control) seedlings given the clipping treatment in 1999
(mean 6 s.e., n = 7). Seedlings were grown under 40 and 3 % of full light. TNC is divided into starch (shaded) and other sugars (open). The graphs
show TNC concentration in cotyledons (A), TNC pools in cotyledons (B), TNC concentrations in roots (C) and TNC pools in roots (D).
Kabeya and Sakai Ð Roots and Cotyledons as Storage Organs in Quercus crispula
543
F I G . 7. TNC concentrations and pools in cotyledons and roots of the initial, control and resprouting seedlings given the clipping treatment in 2001.
Means and s.e. are shown as columns with error bars. Sample sizes were six in initial and control, eight in resprouting at 3-week clipping and ®ve in
6-week clipping. Seedlings were grown under 3 % of full light, and their shoots were clipped 3 and 6 weeks after shoot emergence. TNC is divided
into starch (shaded) and other sugars (open). The graphs show TNC concentration in cotyledons (A), TNC pools in cotyledons (B), TNC
concentrations in roots (C) and TNC pools in roots (D).
roots, were used for resprouting in Q. crispula seedlings, at
least when their shoots were destroyed during the developmental period. The amount of carbohydrate in cotyledons of
control (unclipped) seedlings also decreased during this
period (Fig. 6), but this decrease is considered to have been
due to consumption during the growth of the original shoots
(see Fig. 1B). This hypothesis is supported by the ®nding
that carbohydrates in cotyledons of unclipped seedlings
tended to decrease more than those of resprouting seedlings,
particularly in 3 % light (Fig. 6): larger shoots of unclipped
seedlings would require more reserves for development than
the shoots of resprouting seedlings.
It is still unclear when the storage function of roots
replaces that of cotyledons, but as much carbohydrate was
present in the roots as in the cotyledons from 4 weeks after
shoot emergence onwards. In the 6-week clipping experiment, only a weak tendency was found for the carbohydrate
reserves to decline, in both cotyledons and roots after
resprouting, but this was probably mainly because the
samples were too small to yield statistically signi®cant
results (Fig. 7). Since cotyledons of Q. crispula have
withered and fallen by the second growing season
(D. Kabeya, pers. obs.), the storage role of cotyledons is
presumably replaced by roots within 1 year after germination.
Storage in roots during shoot development and its ecological
signi®cance
Regardless of the light environment, there were already
notable amounts of carbohydrates in roots of Q. crispula
seedlings when their leaf expansion phase started (Fig. 3).
Since the root growth was virtually complete at that time,
particularly in 3 % light (Fig. 1), this nonstructural
carbohydrate is considered to be a reserve substance rather
than a resource used for immediate root growth. Furthermore, most of these carbohydrates are believed to originate
from seed resources. This is because it is unlikely that
seedlings will be able to generate suf®cient amounts of
photosynthates to support growth and development until
their shoot becomes functional and, even if they can
photosynthesize, almost all of the photosynthates are used in
the developing shoots (Dickson et al., 2000). Therefore, we
conclude that some of the seed resources were translocated
to roots and stored as reserves in an early period of seedling
development.
Translocation of reserve substances from seeds to roots
could have an adaptive value as a means of hedging risks.
Hypogeous cotyledons of various species are often eaten by
predators, such as jays and rodents (Bossema, 1979; Forget,
1992; Hoshizaki et al., 1997; Sork, 1987), and this kind of
predation also occurs in Q. crispula (pers. obs.). In addition,
loss of cotyledons often occurs during seedling development. Thus, the risk of seedlings losing carbohydrate
reserves would be likely to increase cumulatively with
time, if all the resources were contained in cotyledons.
Therefore, translocation of some of these resources to roots
in early periods of establishment may reduce the risk of the
plants losing all their reserves through loss of their
cotyledons.
A previous study showed that 80 % of Q. crispula
seedlings, which stored about 20 mg of carbohydrates in
544
Kabeya and Sakai Ð Roots and Cotyledons as Storage Organs in Quercus crispula
their roots, successfully resprouted (Kabeya et al., 2003). In
this study, 20 % of the carbohydrates, in terms of
concentration (Fig. 3), and 20±50 mg in terms of pools
(Fig. 4), were found in roots of the seedlings that had just
started their leaf expansion phase (2 weeks after shoot
emergence). This ®nding indicates that Q. crispula seedlings, whose shoots are destroyed after they have started leaf
expansion, are able to resprout even if their cotyledons are
removed. This hypothesis should be tested in cotyledon
removal experiments in the future.
The decline in carbohydrate reserves in the seedlings'
cotyledons during shoot development showed very similar
trends in both 40 and 3 % light (Figs 3 and 4). If reserves in
cotyledons were needed for growth or survival under low
light, they would be consumed in large amounts and/or
exhausted earlier in seedlings growing under low light
conditions than in counterparts growing under favourable
light conditions, as reported by Jones (1959). Hence, the
results presented here suggest that, in Q. crispula seedlings,
cotyledon reserves do not contribute greatly to survival
under low light conditions. This seems to con¯ict with the
prediction that the reserves in large seeds are likely to be
more important for the survival of seedlings growing under
low light conditions than for similar seedlings growing in
more favourable light (Fenner, 1987; Crow, 1988; Leishman
and Westoby, 1994). However, it is likely that some seed
resources that are translocated to roots play an important
role in survival under such conditions. Quercus crispula
seedlings appear to survive under heavily shaded conditions
by consuming carbohydrate reserves in their roots (Kabeya
et al., 2003). Since they cannot photosynthesize suf®ciently
to maintain themselves under such conditions (Hashimoto
and Shirahata, 1995), seed reserves are expected to act as an
important source of carbohydrates in the roots of established
seedlings in shaded conditions.
CONCLUSIONS
The hypogeal cotyledons of Quercus crispula act as storage
organs during the developmental stage, and the carbohydrate reserves stored in the cotyledons are important for
resprouting after shoot removal. The roots of Q. crispula
seedlings also have carbohydrate reserves, which are
translocated from the cotyledons in the early phase of
development. The amount of carbohydrate stored in roots
increases with growth, and overtakes that in the cotyledons
2 weeks after the growth of functional shoots. However, no
conclusive evidence was found for the hypothesis that
carbohydrate reserves in roots play an important role in
resprouting during the shoot developmental period.
ACKNOWLEDGEMENTS
We thank Akiko Sakai and Kiyoshi Matsui for helpful
suggestions during this study, Drs Elizabeth A. Newell and
Consuelo Bon®l for their constructive comments, Kenichi
Sato for plant cultivation, and Koji Yonekura for providing
us with facilities in the Hakkoda Botanical laboratory.
L I T E RA TU R E C I TE D
Abrams MD. 1996. Distribution, historical development and
ecophysiological attributes of oak species in the eastern United
States. Annales des Sciences Forestieres 53: 487±512.
Andersson C, Frost I. 1996. Growth of Quercus robur seedlings after
experimental grazing and cotyledon removal. Acta Botanica
Neerlandica 45: 85±94.
Bon®l C. 1998. The effects of seed size, cotyledon reserves, and herbivory
on seedling survival and growth in Quercus rugosa and Q. laurina
(Fagaceae). American Journal of Botany 85: 79±87.
Bossema I. 1979. Jays and oaks: an eco-ethological study of a symbiosis.
Behaviour 70: 1±117.
Brookes PC, Wigston DL, Bourne WF. 1980. The dependence of
Quercus robur and Quercus petrata seedlings on cotyledon
potassium, magnesium, calcium, and phosphorous during the ®rst
year of growth. Forestry 53: 167±177.
Callaway RM. 1992. Morphological and physiological responses of three
California oak species to shade. International Journal of Plant
Sciences 153: 434±441.
Canham CD, Berkowitz AR, Kelly VR, Lovett GM, Ollinger SV,
Schnurr J. 1996. Biomass allocation and multiple resource
limitation in tree seedlings. Canadian Journal of Forest Research
26: 1521±1530.
Canham CD, Kobe RK, Latty EF, Chazdon RL. 1999. Interspeci®c and
intraspeci®c variation in tree seedling survival: effects of allocation
to roots versus carbohydrate reserves. Oecologia 121: 1±11.
Crow TR. 1988. Reproductive mode and mechanisms for self-replacement
of northern red oak (Quercus rubra) ± a review. Forest Science 34:
19±40.
Dalling JW, Harms KE. 1999. Damage tolerance and cotyledonary
resource use in the tropical tree Gustavia superba. Oikos 85: 257±
264.
Dalling JW, Harms KE, Aizprua R. 1997. Seed damage tolerance and
seedling resprouting ability of Prioria copaifera in Panama. Journal
of Tropical Ecology 13: 481±490.
Dickson RE, Tomlinson PT, Isebrands JG. 2000. Partitioning of current
photosynthate to different chemical fractions in leaves, stems, and
roots of northern red oak seedlings during episodic growth. Canadian
Journal of Forest Research 30: 1308±1317.
Dubois M, Gilles KA, Hamilton PA, Rebers PA, Smith F. 1956.
Colorimetric method for determination of sugars and related
substances. Analytical Chemistry 28: 350±356.
Fenner M. 1987. Seedling. New Phytologist 106 (Suppl.): 35±47.
Forget P-M. 1992. Seed removal and seed fate in Gustavia superba.
(Lecythidaceae). Biotropica 24: 408±414.
Frost I, Rydin H. 1997. Effects of competition, grazing and cotyledon
nutrient supply on growth of Quercus robur seedlings. Oikos 79: 53±
58.
Grime JP, Jeffrey DW. 1965. Seedling establishment in vertical gradients
of sunlight. Journal of Ecology 53: 621±642.
Hasegawa S. 1984. Basic studies on the conservation of natural coastal
forests in Hokkaido. Research Bulletin of the College Experiment
Forests, Hokkaido University 41: 312±422.
Hashimoto R, Shirahata M. 1995. Comparative study of leaf carbon gain
in saplings of Thujopsis dolabrata var. hondai and Quercus
mongolica var. grosseserrata in a cool-temperate deciduous forest.
Ecological Research 10: 53±64.
Hoshizaki K, Suzuki W, Sakai S. 1997. Impacts of secondary seed
dispersal and herbivory on seedling survival in Aesculus turbinata.
Journal of Vegetation Science 8: 735±742.
Janzen DH. 1976. Reduction of Mucuna andreana (Legminosae) seedling
®tness by arti®cial seed damage. Oecologia 94: 356±360.
Jones EW. 1959. Quercus L. Biological ¯ora of the British Isles. Journal
of Ecology 47: 169±222.
Kabeya D. 2001. Adaptive signi®cance of carbohydrate storage on
resprouting ability in seedlings of Quercus crispula, a deciduous
broad-leaved tree. PhD Dissertation, Tohoku University, Sendai.
Kabeya D, Sakai, A, Matsui, K, Sakai, S. 2003. Resprouting ability of
Quercus crispula seedlings depends on the vegetation cover of their
microhabitats. Journal of Plant Research, 116: 207±216.
Kikuzawa K. 1983. Leaf survival of woody plants in deciduous broad-
Kabeya and Sakai Ð Roots and Cotyledons as Storage Organs in Quercus crispula
leaved forests. 1. Tall trees. Canadian Journal of Botany 61: 2133±
2139.
Kitajima K. 1996. Cotyledon functional morphology patterns of seed
reserve utilization and regeneration niches of tropical tree seedlings.
In: Swaine MD ed. The ecology of tropical forest tree seedlings. New
York: Parthenon Publishing Group, 193±210.
Kruger EL, Reich PB. 1993. Coppicing affects growth, root: shoot
relations and ecophysiology of potted Quercus rubra seedlings.
Physiologia Plantarum 89: 751±760.
Larsen DR, Johnson PS. 1998. Linking the ecology of natural oak
regeneration to silviculture. Forest Ecology and Management 106:
1±7.
Leishman MR, Westoby M. 1994. The role of large seed size in shaded
conditions: experimental evidence. Functional Ecology 8: 205±214.
Matsubara T, Hiroki S. 1985. Ecological studies on the plants of
Fagaceae. IV. Reserve materials in roots and growth until ®ve years
old in Quercus variabilis. Japanese Journal of Ecology 35: 329±336.
Ono K, Terashima I, Watanabe A. 1996. Interaction between nitrogen
545
de®cit of a plant and nitrogen content in the old leaves. Plant and
Cell Physiology 37: 1083±1089.
Rao PB. 1988. Effects of environmental factors on germination and
seedling growth in Quercus ¯oribunda and Cupressus torulosa tree
species of central Himalaya India. Annals of Botany 61: 531±540.
Sakai A, Sakai S, Akiyama F. 1997. Do sprouting tree species on erosionprone sites carry large reserves of resources? Annals of Botany 79:
625±630.
SAS1989. SAS/STAT User's guide (var. 6.0). Cary, NC: SAS Institute.
SAS1998. StatView (var. 5´2). Cary, NC: SAS Institute.
Sokal RF, Rohlf FJ. 1995. Biometry, 3rd edn. New York: Freeman.
Sonesson LK. 1994. Growth and survival after cotyledon removal in
Quercus robur seedlings, grown in different natural soil types. Oikos
69: 65±70
Sork VL. 1987. Effects of predation and light on seedling establishment in
Gustavia superba. Ecology 68: 1341±1350
Westoby M, Jurado E, Leishman MR. 1992. Comparative evolutionary
ecology of seed size. Trends in Ecology and Evolution 7: 358±372.