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. 2005 Jul 1;96(3):479–488. doi: 10.1093/aob/mci200

The Relative Importance of Carbohydrate and Nitrogen for the Resprouting Ability of Quercus crispula Seedlings

DAISUKE KABEYA 1,*, SATOKI SAKAI 2
PMCID: PMC4246782  PMID: 15994839

Abstract

• Background and Aims Plants need some kind of stored resources to resprout after shoot destruction. The aim of this study was to determine the relative importance of carbohydrate and nitrogen (N) storage levels for their ability to resprout.

• Methods A shoot clipping experiment was conducted on Quercus crispula seedlings, which were grown in a factorial experimental design, with two light levels (40 % and 3 % of full light) and three nutrient concentrations (low, medium and high).

• Key Results At the time of shoot clipping (the end of spring leaf expansion), seedlings exposed to 40 % light had an average total non-structural carbohydrate (TNC) concentration of 17·0 % in their roots compared with 4·9 % in the roots of seedlings exposed to 3 % light, and the average amount of TNC (TNC pools) in the roots was 203·8 mg and 20·0 mg at 40 % light and 3 % light, respectively. In contrast, root N concentration averaged 2·3 % in the 3 % light treatment compared with 1·2 % in the 40 % light treatment, and it increased with successive rises in nutrient concentrations at both light levels. Regardless of the nutrient status, at the 40 % light level >80 % of the seedlings resprouted after shoot clipping. Few seedlings, however, resprouted at the 3 % light level, particularly in the medium- and high-nutrient treatments. Furthermore, both root TNC concentrations and TNC pools decreased after resprouting, but the amount of root N remained constant.

• Conclusions These results suggest that carbohydrate storage has a stronger influence on resprouting in Quercus crispula than N storage. However, the size of the resprouting shoot was positively correlated with the amount of both N and TNC in roots. The level of N storage is, therefore, also important for the growth of resprouting shoots.

Keywords: Quercus crispula, seedling, resprouting, light availability, nutrient availability, carbohydrate, nitrogen, total non-structural carbohydrate

INTRODUCTION

In woody plants, it is generally believed that stored carbohydrates are the most important resources controlling resprouting after shoot destruction (Bond and Midgley, 2001). Since shoot destruction causes the loss of photosynthetic tissues (leaves), other carbon sources are required, at least until new shoots become functional (i.e. until they are able to photosynthesize sufficiently). This conclusion is supported by data showing remarkably high levels of starch storage in resprouter plants, which regenerate by resprouting after shoot destruction, for example, following fire (Pate et al., 1990; Bell et al., 1996; Bell and Ojeda, 1999; Bell, 2001; Verdaguer and Ojeda, 2002). It has been recorded that carbohydrate levels decrease more than those of any other nutrients in storage organs at the time of resprouting (Jones and Laude, 1960; Miyanishi and Kellman, 1986; Canadell and López-Soria, 1998). Other studies have also found that resprouting ability (i.e. the likelihood of resprouting and/or the growth rate of the resprouting shoot) depends on the level of carbohydrate storage prior to shoot destruction (Kays and Canham, 1991; Bowen and Pate, 1993; McPherson and Williams, 1998; Sakai and Sakai, 1998; Kabeya et al., 2003). There are some tree species, however, whose resprouting abilities are not related to the level of carbohydrate storage (see, for example, Taylor and Pharis, 1982; Garcia et al., 2001; Cruz et al., 2003).

Furthermore, in some forage species, nitrogen storage is more important for regrowth after defoliation than carbohydrate storage (Volenec et al., 1996). Such species store nitrogen as amino acids or vegetative storage protein in their taproots, stolons and stubble (i.e. remaining shoots). This stored nitrogen is used in the regrowth of new shoots (Ourry et al., 1994). Recently, El Omari et al. (2003) showed that the resprouting capacity of Quercus ilex seedlings was inhibited when they were grown under N-limited conditions, even when they had accumulated high levels of non-structural carbohydrates (TNC) in their roots.

In other tree species it is, therefore, possible that the availability of nutrients, particularly nitrogen, may influence resprouting after shoot destruction. Nitrogen may be especially important in temperate and wet regions where it is often one of the most limiting nutrients (Kaye and Hart, 1997; Fenn et al., 1998). In both forage and tree species, foliage is the main nitrogen sink. Hence, the loss of shoots results in the loss of nitrogen, so both carbohydrate and nitrogen supplies are required for shoot regrowth. It is predicted, therefore, that depletion of the nitrogen supply could result in the failure of trees to resprout.

It is important to determine whether carbohydrate or nitrogen has the major influence on resprouting ability. The C/N balance theory suggests that the levels of total non-structural carbohydrate (TNC) and nitrogen within plants will exhibit inverse responses to the environmental conditions (cf. Chapin et al., 1990). That is, when nutrient availability limits their growth plants tend to accumulate an excess of carbon in the form of TNC and their nutrient levels become relatively low, while under carbon limitation their nutrient levels rise. Hence, by testing the resprouting ability of seedlings in experiments with light × nutrient factorial designs, it should be possible to clarify whether levels of TNC or nitrogen are more important for resprouting.

In addition, nitrogen availability may directly affect the ability to resprout. In contrast to carbohydrate, the assimilation of which is stopped by shoot destruction, plants can continue to absorb nutrients via their roots. Although defoliation limits or suppresses the acquisition and assimilation of nitrogen in grass (Volenec et al., 1996), there are contradictory results for trees (e.g. Lovett and Tobiessen, 1993; Millard et al., 2001). The absence of a decrease in nitrogen in the remaining plant parts at the time of resprouting observed by authors such as Miyanishi and Kellman (1986) suggests that absorption of nutrients can continue even when all shoots are removed. Therefore, both endogenous and exogenous sources of nitrogen need to be considered for resprouting after shoot destruction.

Quercus crispula, a deciduous tree species common throughout the cool-temperate forests of Japan, is relatively tolerant to disturbance (Kamitani, 1986; Shibuya et al., 1997). Seedlings are often damaged by various types of disturbance, but they can resprout after shoot destruction (Kanazawa, 1983). They store large amounts of carbohydrate in their taproots, particularly under favourable light conditions, and their resprouting ability depends on the size of their carbohydrate reserves (Kabeya et al., 2003). However, the relative importance of the levels of carbohydrate and nitrogen storage (or availability) to the resprouting ability of this species is still unclear.

In this study, the aim was to determine how the availability of nitrogen and light, the storage of nitrogen and carbohydrate, and their interactions, affect the resprouting ability of Q. crispula seedlings. For this purpose, 1-year-old Q. crispula seedlings, grown in a factorial design with two light and three nutrient treatments, were used. Then seedling growth, the levels of carbohydrate and nitrogen storage and resprouting ability after shoot clipping were measured.

MATERIALS AND METHODS

Seedling growing conditions

The experiments were conducted at the Hakkoda Botanical Laboratory (40°38′N, 140°51′E, 900 m a.s.l.) between 1999 and 2000. The acorns of Q. crispula that were used in this experiment were collected from the eastern side of Lake Towada (40°26′N, 140°56′E, 400 m a.s.l.) in the autumn of 1998. The acorns were washed and stored in a dark cold-room (<5°C) until planting.

On 15 June 1999, approx. 300 acorns were sown in Akadama soil:sand (2 : 1, v/v) in 12 × 14 cm pots with a single acorn per pot. The planted pots were divided into two groups, one of which was placed in 40 % of full light and the other in 3 % of full light (in both cases, varying between 1642 and 2248 µmol m−2 s−1 on different days), using shade cloths to control the light levels. Within each light treatment, the pots were divided into three groups, each of which was given a different nutrient treatment. The first group received no fertilizer (low-nutrient treatment), while the second and third groups received fertilizer containing 10 % N, 10 % P and 10 % K by weight (Pro-long-total 70, Asahi Kagaku Co., Japan) twice during each growing season (once at the start and once in the middle). Fertilizer we applied at the rate of 2 g plant−1 to the second group (medium-nutrient treatment) and 4 g plant−1 to the third group (high-nutrient treatment). Seedlings were grown from June 1999 to September 2000. In total, 246 seedlings were used in the experiments.

Biomass measurement

There were three harvests: at the end of the first growing season (12 October 1999, Harvest 1); at the start of the second growing season, at which time almost all seedlings had already completed their leaf expansion (26 June 2000, Harvest 2); and at the end of the second growing season (26 September 2000, Harvest 3).

At harvest, seven seedlings were selected randomly from each treatment. They were collected in the morning, separated into leaves, stems and roots, and, within 6 h of collection, these fractions, were placed in an oven where they were dried at 70°C for 3 d, then weighed. Hereafter, these seedlings are referred to as control seedlings to distinguish them from the shoot-clipped seedlings (see below).

Clipping treatment

Clipping was performed to assess the effect of light and nutrient availabilities and the level of carbohydrates and nitrogen storage on the resprouting ability of the seedlings. On 26 June 2000 (the same day as Harvest 2), 20 seedlings were selected randomly from each of the six treatments (two light levels × three nutrient levels), and their shoots were clipped down to 1·5 cm above the cotyledonary node. There were at least three dormant buds on the stem below the clipping point (two at the cotyledonary node and one or more on other parts of the stem). Clipped shoots were divided into leaves and stems, dried in an oven (70°C) for 3 d, and weighed. From the weight of the clipped shoots, the root weight of shoot-clipped seedlings was estimated using the allometric relationship between root weight and shoot weight of the control seedlings.

Clipped seedlings were checked every 2 d until the sixth week after clipping to determine whether they had resprouted. The date of bud break (i.e. the start of resprouting) and the date that leaf expansion began were recorded; seedlings for which these dates were not recorded were excluded from all analyses except those concerning the number of resprouting seedlings. Resprouted seedlings were harvested on seven occasions: 23 July, 29 July, 5 August, 12 August, 29 August and 5 September. At each harvest, seedlings whose leaf expansion had begun but had not been completed were sampled.

The collected seedlings were separated into resprouted shoots (further divided into leaves and stems), residual (original) stems and roots, and placed in an oven within 6 h of harvest. These fractions were dried at 70°C for 3 d and weighed. After that, root TNC concentrations as well as root and resprouted shoot N concentrations were determined, as described below.

Carbohydrates and nitrogen analysis

At Harvests 1 and 3 only the amounts of TNC in the roots of the control seedlings were determined. In addition to the TNC amounts in the roots, the amounts of nitrogen in roots and leaves of the control seedlings were determined at Harvest 2. The amounts of TNC in the roots and those of nitrogen in the roots and shoots of resprouted seedlings in the clipping experiment were also determined.

Roots and leaves of the weighed samples were ground using a Wiley mill, until the residue would pass through a no. 65 mesh screen; this was dried in the oven for a further day prior to carbohydrate and nitrogen analysis. Nitrogen contents in roots and leaves were determined using an N/C analyser (NC-80, Shimadzu, Japan). The concentration of carbohydrate in roots was analysed following the method described in Kabeya et al. (2003), in which starch in the sample was hydrolysed to glucose with amyloglucosidase (Sigma), and the total amount of all sugars (hydrolysed starch and other soluble sugars; TNC) was measured using the phenol–sulphuric acid method. TNC and N pools, i.e. the total amounts of TNC and N, were calculated by multiplying the concentration of TNC and N, respectively, by the dry mass.

Statistical analysis

Because there were positive correlations between the means and variances of almost all data sets, it was concluded that the gamma distribution was more appropriate for estimating the error distribution than the normal distribution. Hence, a generalized linear model for a likelihood ratio type 3 analysis, in which the gamma distribution approximated the error distribution, was used for testing the effects of light and nitrogen availability on each plant size parameter. For the average amount of TNC (TNC pools) in roots at Harvest 2, however, Wald type 3 analysis was used instead of likelihood ratio analysis, because the likelihood ratio statistic could not be calculated. In the clipping experiment, the effect of resprouting on TNC and nutrient levels of the seedlings was also tested using this procedure. In this analysis ‘environmental conditions’ were used as a main factor with four categories: low-nutrient, medium-nutrient, high-nutrient at 40 % light and low-nutrient at 3 % light. The sample size was insufficient for statistical analysis of the effects of the medium- and high-nutrient treatments at the 3 % light level (see Results). A pair-wise randomization test was conducted, using Holms' method to adjust for type I errors (Sokal and Rohlf, 1995) for multiple comparisons between means of each parameter. The logit model was used to test the effects of light/nitrogen availabilities and the level of TNC/nitrogen on the number of resprouting seedlings. Correlation and partial correlation was used to analyse the relationships between resprouted shoot mass, estimated root mass at the time of clipping (initial root mass) and TNC and nitrogen pools in roots at the time of clipping (initial TNC pools and initial N pools, respectively). The initial TNC and N pools in the roots of the clipped seedlings were calculated from the estimated initial root mass of the clipped seedlings and the mean concentration of TNC (or nitrogen) in the roots of the control seedlings at Harvest 2. SAS/STAT software (version. 6·1·1; SAS Institute, Inc.) was used for all statistical analyses except the randomization tests.

RESULTS

The effects of light and nutrient availabilities on control seedlings

At the end of the first growing season (Harvest 1), the total dry mass of the low nutrient (control) seedlings in 40 % light conditions averaged 2·46 ± 0·31 g and was significantly greater than that of the control seedlings growing under 3 % light conditions, which averaged 0·97 ± 0·16 g (Table 1). Indeed, throughout the experimental period, the mean total dry mass of the control seedlings at 40 % full light was greater than that of seedlings growing in 3 % light, particularly in the medium- and high-nutrient treatments (Table 1). Under both light conditions, nutrient availability only affected total dry mass at the end of the second growing season (Harvest 3, Table 2); the total dry mass of the medium- and high-nutrient seedlings was greater than that of the low-nutrient seedlings, but these values were not significantly different (Table 1).

Table 1.

Total dry mass and TNC concentration in the roots of Quercus crispula seedlings harvested at the end of the first growing season (Harvest 1) and the start and end of the second growing season (Harvests 2 and 3, respectively)

Harvest 1
 Harvest 2
 Harvest 3
Light
 Nutrient
 Mean
 s.e.
 Mean
 s.e.
 Mean
 s.e.
Total drymass (g plant−1) 40 % L 2·46 0·31a 1·44 0·24a 3·53 0·32b
M 2·11 0·14a 2·52 0·34a 9·25 0·65a
H 2·74 0·41a 2·45 0·28a 10·00 0·89a
3 % L 0·97 0·16a 0·83 0·11a 1·38 0·25a
M 1·14 0·13a 0·89 0·14a 2·23 0·28a
H 1·22 0·26a 0·64 0·16a 2·01 0·27a
Root TNC concentration roots (%) 40 % L 43·6 1·7a 28·6 3·3a 45·3 1·7a
M 35·1 1·7b 11·1 2·9b 29·5 1·3b
H 35·3 2·6b 11·4 3·1b 29·4 2·4b
3 % L 29·5 2·1a 5·5 1·2a 45·6 2·0a
M 28·9 1·8a 5·6 2·3a 24·7 1·7b
H 23·4 2·4a 3·3 0·5a 25·9 2·5b
Root TNC pool (mg plant−1) 40 % L 786·2 118·8a 311·7 72·3a 1099·7 91·7a
M 495·5 49·6a 153·7 40·9a 1421·4 244·1a
H 603·8 87·5a 145·9 36·7a 1339·5 149·4a
3 % L 164·6 36·4a 27·8 9·6a 356·5 68·7a
M 165·8 27·1a 22·5 8·9a 226·3 41·2a
H 143·4 43·7a 9·6 3·5a 215·4 42·7a
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Within each light treatment values with different letters are significantly different at P < 0·05 between the nutrient availability (pair-wise randomization tests were used in each comparison and type I errors were adjusted by Holms' method).

Table 2.

Likelihood ratio statistics with probabilities for type 3 analysis in the generalized linear model to test the influence of light availability (Light) and nutrient availability (Nutrient) on the total dry mass, TNC concentrations and nitrogen concentrations of the seedlings

χ2 value (P)
Light (1*)
 Nutrient (2*)
 L × N (2*)
Total dry mass Harvest 1 28·6 (<0·001) 1·5 (0·480) 1·1 (0·590)
Harvest 2 30·4 (<0·001) 2·2 (0·340) 3·4 (0·180)
Harvest 3 65·6 (<0·001) 20·1 (<0·001) 1·1 (0·580)
% TNC in roots Harvest 1 28·3 (<0·001) 10·4 (0·010) 3·7 (0·160)
Harvest 2 23·3 (<0·001) 4·3 (0·110) 2·2 (0·340)
Harvest 3 4·5 (0·030) 49·4 (<0·001) 3·6 (0·170)
TNC pools in roots Harvest 1 41·9 (<0·001) 1·6 (0·448) 1·5 (0·473)
Harvest 2† 24·6 (<0·001) 5·3 (0·069) 4·3 (0·118)
Harvest 3 67·8 (<0·001) 1·1 (0·585) 6·8 (0·033)
% N in roots Harvest 2 84·2 (<0·001) 92·7 (<0·001) 39·4 (<0·001)
% N in leaves Harvest 2 59·6 (<0·001) 50·2 (<0·001) 17·9 (<0·001)
N pools in roots Harvest 2 6·3 (0·010) 19·2 (<0·001) 1·9 (0·390)
N pools in leaves Harvest 2 0·1 (0·770) 23·5 (<0·001) 10·0 (0·010)
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Seedlings were grown under two light (40 % and 3 %) and three nutrient (low, medium and high) conditions and there were three harvests.

*

Degrees of freedom.

†

Wald statistics for type 3 analysis.

Under the 40 % light conditions, seedlings stored high concentrations of TNC in their roots (Table 1). Since large quantities of TNC in the roots were utilized during spring growth, root TNC concentrations declined between Harvests 1 and 2. Under the 40 % light conditions, however, 28·6 ± 3·3 %, 11·1 ± 2·9 % and 11·4 ± 3·1 % TNC were still observed at Harvest 2 in seedlings from the low-, medium-, and high-nutrient treatments, respectively (Table 1). Consequently, the TNC concentration in roots was significantly higher under the 40 % light than under the 3 % light conditions throughout the experiment (Table 2). At 40 % light, seedlings in the low-nutrient treatment had higher concentrations of TNC in their roots than those in the medium- and high-nutrient treatments at the end of both seasons (Harvests 1 and 3, Table 1).

TNC pools in roots of the seedlings grown under 40 % light and 3 % light averaged 145·9 ± 36·7 to 1421·4 ± 244·1 mg and 9·6 ± 3·5 to 356·5 ± 68·7 mg, respectively, in the various treatments (Table 1), and the root TNC pools of the former were significantly larger than those of the latter at every harvest (Table 2). Compared with the effect of light condition, the effect of nutrient availability on root TNC pools was small (Table 2) and there was little difference in root TNC pools between any of the nutrient treatments, under either the 40 % or 3 % light conditions (Table 1).

At Harvest 2, the mean nitrogen concentrations in both roots and leaves were affected by the light and nutrient availabilities, and interactions between treatments were also found (Table 2). Leaf N concentrations ranged from 2·3 to 3·9 % under the 40 % light and from 4·1 % to 5·4 % under the 3 % light conditions (Table 3), and they were lower under the former than the latter for every nutrient treatment (P < 0·001 pair-wise comparison at all nutrient treatments). For both light treatments, leaf N concentrations of low-nutrient seedlings were lower than those of medium- and high-nutrient seedlings. The nitrogen concentration in roots ranged from 0·6 % to 1·7 % at 40 % light and from 1·4 % to 3·1 % at 3 % light (Table 3). Root N concentration was higher at 3 % light than at 40 % light in all nutrient treatments (P < 0·001 for all nutrient availabilities), and showed successive increases with successive increases in nutrient availability for both light treatments. At 40 % light, N pools in roots and leaves were lower for the low-nutrient treatment than for the medium- and high-nutrient treatments (Table 3). At 3 % light, on the other hand, N pools in roots and leaves were not significantly different between the nutrient treatments (Table 3). Across all the treatments, the root N concentration was lower than in the leaves (Wilcoxon sign rank test; P < 0·001), but there was no significant difference between the root N pool and the leaf N pool (P = 0·64).

Table 3.

N concentrations and pools in leaves and roots of Q. crispula seedlings collected at Harvest 2

Leaves
 Roots
Light
 Nutrient
 Mean
 s.e.
 Mean
 s.e.
N concentration (%) 40 % L 2·3 0·1b 0·6 0·0c
M 3·8 0·2a 1·4 0·0b
H 3·9 0·2a 1·7 0·1a
3 % L 4·1 0·2b 1·4 0·1c
M 5·4 0·4a 2·4 0·1b
H 5·2 0·2a 3·1 0·2a
N pool (mg plant−1) 40 % L 3·9 0·8b 6·2 1·0b
M 21·6 3·6a 21·8 3·6a
H 19·1 2·5a 22·3 2·1a
3 % L 6·7 0·8a 5·9 0·8a
M 10·6 1·9a 10·4 1·6a
H 7·6 2·0a 8·6 2·0a
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Within each light treatment values with different letters are significantly different at P < 0·05 (pair-wise randomization tests were used in each comparison and type I errors were adjusted by Holms' method).

Clipping treatment

After clipping, >80 % of the seedlings (17, 18 and 16 out of 20 seedlings in the low-, medium- and high-nutrient treatments, respectively) resprouted in 40 % of full light. In contrast, few seedlings resprouted under 3 % light; six, one and zero out of 20 seedlings, respectively. When the numbers of medium-nutrient and high-nutrient seedlings were combined, light availability was found to have a significant effect on the probability of resprouting (Logit model, χ2 = 39·1, P < 0·001) and a significant interaction between light and nutrient availability was found (χ2 = 5·9, P = 0·016), indicating that nutrient availability affected the probability of resprouting only in 3 % light (Fishers' exact test, P = 0·013). Across all treatments there was a significant positive relationship between the percentage of resprouted clipped seedlings and root TNC concentrations at the time of clipping (Harvest 2) (logistic regression, R2 = 0·26, P < 0·001, Fig. 1A). Furthermore, a negative correlation between TNC and N concentrations in the roots (r = −0·62, P < 0·001, Fig. 2) resulted in a significant negative relationship between the resprouting percentage and the root N concentration at the time of clipping (R2 = 0·26, P < 0·001, Fig. 1B).

Fig. 1.

Fig. 1.

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Relationship between the probability of resprouting after shoot clipping and initial TNC (A) and N (B) concentrations in the roots (TNC and N concentrations in roots of control seedlings at Harvest 2). Dashed lines show the results of logistic regressions; logit(y) = −2·21 + 0·23 × (TNC concentration) (A) and logit(y) = 3·73–2·19 × (N concentration) (B). Where logit(y) = log[y/(1 – y)]. The symbols show the resprouting percentage and the mean TNC and N concentrations for each of the treatments, as indicated by the key in A.

Fig. 2.

Fig. 2.

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The relationship between N concentration and TNC concentration in the roots of Q. crispula seedlings. For a key to the symbols, see Fig. 1A.

Compared with concentrations at the time of clipping, TNC concentrations in the roots of the seedlings in low-nutrient conditions at 40 % light declined after resprouting (from 28·6 ± 3·3 % to 21·6 ± 2·1 %), and similar trends were detected for both the medium- and high-nutrient conditions (Fig. 3A). In total, the decline was statistically significant (Table 4, resprouting). TNC pools in the roots also showed the same trends as TNC concentrations (Fig. 3B). However, an interaction was found between resprouting and condition (Table 4), and a significant decline was found only in high-nutrient conditions at 40 % light. Compared with the TNC concentration, the N concentration in roots changed less after resprouting (Fig. 3B). In low-nutrient conditions at 40 % light, for example, the root N concentration was 0·6 ± 0·0 % and 0·7 ± 0·1 % before and after resprouting, respectively. Furthermore, N concentrations in the roots increased significantly after resprouting (Table 4). This appears to have been due to the reduction in root TNC (Fig. 3C), since the N percentages of structural root weight (root dry mass – TNC pools in roots) at the time of clipping and after resprouting were not significantly different (Table 4). Neither root N pools, nor the total amount of N before and after resprouting were significantly different (Fig. 3D and Table 4).

Fig. 3.

Fig. 3.

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TNC concentrations (A), TNC pools (B), nitrogen concentrations (C) and nitrogen pools (D) (means and standard error) of Q. crispula seedlings at the time of shoot clipping (control) and after resprouting in each treatment. Although seedlings were grown in two light (40 % and 3 % of full light) and three nutrient (low, medium and high) conditions, the data for the medium- and high-nutrient treatments in 3 % light are not shown because the sample sizes were too small to be statistically significant.

Table 4.

Likelihood ratio statistics with probabilities for type 3 analysis in the generalized linear model to test whether or not the independent values were different between before and after resprouting (Resprouting), the effect of environmental conditions (Condition), and their interaction on the TNC and nitrogen concentrations in roots and the percentage of nitrogen in the structural root mass of the seedlings in the clipping experiment

χ2 value (P)
Resprouting (1*)
 Condition (3*)
 R × C (3*)
Root TNC concentration 5·8 (0·016) 58·1 (<0·001) 5·6 (0·135)
Root TNC pools† 1·6 (0·203) 50·1 (<0·001) 8·4 (0·038)
Root N concentration 4·3 (0·037) 115·7 (<0·001) 7·1 (0·068)
Root N concentration (proportion to structural root weight) 0·3 (0·558) 95·1 (<0·001) 3·6 (0·312)
Root N pools 0·1 (0·704) 66·9 (<0·001) 5·9 (0·118)
Total N pools 0·8 (0·377) 69·8 (<0·001) 6·0 (0·112)
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*

Degrees of freedom.

†

Wald statistics for type 3 analysis.

The mean dry masses of resprouting shoots of the clipped seedlings ranged from 8·9 ± 2·7 mg to 98·8 ± 1·98 mg. Resprouting shoot mass increased with increases in nutrient availability in 40 % light (Fig. 4). Overall, there were significant positive correlations between the initial N pools in the roots and the resprouting shoot mass of the resprouted seedlings (Fig. 5). Moreover, partial correlation analysis detected a significantly positive relationship between the initial N pools in the roots and the resprouting shoot mass and a marginally positive relationship between initial TNC pools in the roots and the resprouting shoot mass (Table 5).

Fig. 4.

Fig. 4.

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Dry mass of the resprouted shoots (separated into leaves and stems) of the seedlings in the clipping experiment (means and standard error). The results of the ANOVA are shown in Table 4. Bars marked with different letters are significantly different at P < 0·05, as determined by a pair-wise randomization test (type I error was adjusted by Holms' method).

Fig. 5.

Fig. 5.

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The relationships between resprouting shoot mass and initial N pools (A), initial TNC pools (B) and initial root mass (C) of Q. crispula seedlings. For a key to the symbols, see Fig. 1A. The correlation coefficient marked with the asterisks is statistically significant at P < 0·001.

Table 5.

Partial correlations for resprouted shoot mass, initial nutrient reserves and initial root mass of the clipped seedlings (with P-values)

 Resprouted shoot mass
 Initial N pools
 Initial TNC pools
 Initial root mass
Resprouted shoot mass –
Initial N pools 0·44 (0·006) –
Initial TNC pools 0·32 (0·050) −0·96 (<0·001) –
Initial root mass −0·32 (0·050) 0·98 (<0·001) 0·98 (<0·001) –
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Initial root mass (root dry mass at the time of clipping) was estimated from the clipped shoot using an allometric relationship. Initial amounts of the resources (N and TNC pools in the roots at the time of clipping) were calculated by multiplying the estimated initial root mass and N and TNC concentrations (respectively) in roots of the control seedlings at Harvest 2.

DISCUSSION

The effects of carbohydrate storage and nutrient availability on resprouting

It was possible to demonstrate that, in Q. crispula seedlings, the level of carbohydrate storage is more important for resprouting after shoot destruction than the level of nitrogen. In particular, whether or not seedlings were able to start resprouting depended on the level of stored carbohydrate. In this study, differences in the level of carbohydrate in roots were largely attributable to differences in light availability. Light availability may, therefore, directly control whether or not plants with destroyed shoots can resprout (e.g. Johansson, 1986). However, Canham et al. (1994) showed that shoot clipping in the winter has little effect on seedling mortality (i.e. seedlings clipped at this time could re-grow shoots) regardless of the whether the seedlings were in high-light environments or in the shade. The high ability to recover from shoot damage in the dormant season could be due to the large amount of carbohydrate stored at this time (see, for instance, Kays and Canham 1991). In addition, it has been found that if Q. crispula seedlings under 3 % light are clipped during their dormant season, when they store carbohydrates in their roots at concentrations similar to those in seedlings under 40 % light (38·7 ± 2·4 % and 36·4 ± 1·4 % of total root weight at 3 % light and 40 % light, respectively), >90 % of them resprout (D. Kabeya, unpubl. res.). Thus, it is concluded that with sufficient carbohydrate reserves, which can be produced with adequate light, there is a high probability of resprouting after shoot destruction in Q. crispula seedlings. This is consistent with many previous studies which have concluded that the level of stored carbohydrate plays an important role in survival after shoot destruction (Robison and Massengale, 1968; Bowen and Pate, 1993; Canadell and López-Soria, 1998; McPherson and Williams, 1998; Canham et al., 1999; Kabeya et al., 2003).

In contrast, the limits to regrowth after disturbance caused by deficiencies of resources other than carbohydrates have not yet been established (but see El Omari et al., 2003), although a number of conjectures on this subject have been made in some publications (e.g. Chapin et al., 1990; Canadell and López-Soria, 1998). If anything, an excess of nitrogen may adversely affect the probability of resprouting in Q. crispula seedlings. Although most of the N-related differences in resprouting ability found were attributed to the negative correlation between carbohydrate and N levels in the roots, the probability of resprouting for low-nutrient seedlings was higher than that for medium- and high-nutrient seedlings in 3 % light, and the present data show that this could not be due to differences in the amount of carbohydrate stored. In some studies, the nitrogen concentration was found to be higher in plants growing under low light than in plants growing under high light because of carbon shortage (Dale and Causton, 1992; Niinemets, 1997; Zhao, 1998). In such carbon-limited environments, a relative excess of nutrients increases seedling mortality (cf. Lambert, 1986; Catovsky and Bazzaz, 2002), and hence, it may prevent the commencement of resprouting in this species.

Resprouting success (the degree of tolerance to disturbance) depends on the ability of the plants to resprout after shoot destruction (the probability of resprouting) and the size the resprouted shoots can attain. As discussed above, the former depends mainly on the level of stored carbohydrate, but the latter seems to depend on the storage levels of both carbohydrate and nitrogen. It is known that high nitrogen availability induces plant regrowth after partial defoliation and that nitrogen in the remaining plant parts is also utilized for reconstructing new shoots when absorption or fixation of external nitrogen is insufficient (see, for instance, Volenec et al., 1996; El Omari et al., 2003). Furthermore, in several plant species, initial resprouting shoot size (initial growth) is also correlated positively with the level of stored carbohydrate (McPherson and Williams, 1998; Kabeya et al., 2003). Whether the strongest influence on regrowth is carbon or nitrogen supply depends on which resource limits the growth of the plant. That is, the initial size of the resprouting shoot depends on the level of stored carbohydrate in roots when light availability (and thus the level of stored carbohydrate) is limited (Kabeya et al., 2003), but it is controlled by nitrogen supply if the seedlings have stored sufficient carbohydrate.

It was found that the nitrogen pools in the roots of the Q. crispula seedlings were as large as those in the leaves. In addition, the root nitrogen concentration increased successively with successive increases in nutrient availability, even though the leaves were saturated with nitrogen. These results suggest that under high nutrient availability Q. crispula seedlings store nitrogen, which is more than is needed for the current needs of their leaves, in their roots. This is consistent with data concerning Q. ilex seedlings (El Omari et al., 2003). Although it was not possible to distinguish between stored nitrogen and structural nitrogen, Bollmark et al. (1999) have shown that in Salix viminalis the level of non-protein N classified as a storage substance increases with increasing nutrient availability. It is considered that, in Q. crispula too, some nitrogen in the roots acts as a storage substance.

El Omari et al. (2003) showed that the Q. ilex seedlings they examined did not directly use soil-absorbed N to create resprouting shoots, but that N stored in the roots was translocated to new shoots during resprouting. In Q. crispula, however, it is still unclear whether endogenous or exogenous N is most important for resprouting. When planning the present experiment, it was expected that root N pools would decrease and the total amount of N would remain constant if the plants used endogenous N (root N) for resprouting, via translocation to resprouting shoots, rather than using exogenous N. If, however, the seedlings could absorb N and use it for resprouting, it was expected that the total amount of N would increase. Although it was demonstrated that the resprouting shoot mass correlated positively with the root N level, neither a significant decrease in the root N pool nor an increase in the total amount of N after resprouting was found. This is mainly because the resprouting shoot was too small to significantly affect the N pool. To detect N fluctuations during resprouting conclusively, a tracer experiment is required.

The effect of nutrient availability on carbohydrate storage

Kabeya et al. (2003) showed that the amount of carbohydrate stored in Q. crispula seedlings increased with an increase in light availability. They concluded, therefore, that the carbohydrate reserves of Q. crispula seedlings are affected by the light environment. However, the level of stored carbohydrate is also affected by nutrient availabilities, particularly nitrogen (Koricheva et al., 1998). That is, plants growing in a nutrient-limited environment contain more carbon than they require, and consequently excess carbon accumulates as stored carbohydrate (Chapin et al., 1990). This carbohydrate accumulation is greater under favourable light conditions. Thus, the large amount of carbohydrate stored in Q. crispula seedlings may be due to the accumulation of carbohydrates in excess to their growth requirements. However, after leaf flush, even in the high-nutrient treatment, where growth was not limited by nitrogen availability, Q. crispula seedlings growing under 40 % of full light stored a greater amount of TNC in their roots than those growing under 3 % light. These findings suggest that carbohydrate storage in the roots of Q. crispula seedlings growing under favourable light conditions reflects not only accumulation caused by nutrient limitation, but also reserve storage as an adaptive mechanism against environmental stress (e.g. shoot destruction), in accordance with data presented by Wyka (2000). Furthermore, it was shown that the level of reserve storage is affected by the environmental conditions, as predicted by Iwasa and Kubo (1997) and Bellingham and Sparrow (2000) in their theoretical analyses.

Conclusions

In Q. crispula seedlings, stored carbohydrate is a more important resource than nitrogen for resprouting, in particular for determining whether or not resprouting can commence after shoot destruction. However, with sufficient carbon, the size of the resprouting shoot is affected by the nitrogen availability in the environment and/or the level of nitrogen stored in the roots. Thus, nitrogen is also an important determinant of the ability of this species to resprout.

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Acknowledgments

We would like to thank Sakai Akiko, Matsui Kiyoshi and Hikosaka Kouki for their many constructive suggestions about this study. Thanks are also due to Sato Kenichi for cultivating the plants, and to Yonekura Koji for our stay at the Hakkoda Botanical Laboratory. The submission of SAS/STAT was performed with the assistance of the Computer Center for Agriculture, Forestry and Fisheries Research, MAFF, Japan.

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