In order to evaluate the degree of carbon autonomy for fruit development, the carbon source-sink relationship in fruit-bearing branchlets of mature deciduous forest trees was manipulated in situ. The tests included half and complete defoliation, girdling or the combination of both treatments, which were applied on fruiting branchlets by using a canopy crane. Concentrations of non-structural carbohydrates (NSC) were analysed in different branchlet tissues and fruits, to identify situations of carbon imbalances induced by the treatments. NSC concentrations of branchlets were generally lower under treatments resulting in decreased fruit growth. All three investigated species (Carpinus betulus, Fagus sylvatica and Tilia platyphyllos) exhibited complete carbon autonomy of fruiting at the level of whole, undisturbed branchlets, since neither a decrease of total infructescence biomass, nor of individual fruit mass occurred on girdled, un-defoliated branchlets. On girdled, 100% defoliated branchlets, fruit biomass relative to controls decreased by approximately 50% in Carpinus and Tilia, but by almost 80% in Fagus, which can be explained by different proportions of photosynthetically active infructescence tissues among the species. In contrast to the other two species, Tilia branchlets did not import carbon to compensate for assimilate loss after defoliation.
Tree crowns may be seen as populations of modules, which, at least in some aspects, can function as autonomous units (Watson 1986). A single branchlet, consisting of a short stem supporting a couple of leaves would be the smallest functional unit. The theory of branch and branchlet autonomy received special attention in tree physiological studies (see Sprugel, Hinckley & Schaap 1991 for an extensive review), because a single branch exhibits many physiological characteristics of a whole tree, namely fixation, transport and storage of photo-assimilates and the possession of a hierarchical population of meristems (buds), while at the same time, a branch is small enough to be experimentally manageable. The concept of branch autonomy, however, is also limited, since all branches are connected to the same trunk and root system and therefore compete for soil nutrients and water (Sprugel 2002).
Undamaged branchlets export carbon during most of the vegetation period, and with regard to carbon, a single, foliated branch could be completely autonomous. However, there may be two particular periods, when branches nonetheless depend on the import of stored carbon from other tree parts (i.e. older branches, stem, roots): (1) at the beginning of the growing season, when carbon demand for growth may exceed the fresh carbon supply by the young, unfolding leaves, particularly in deciduous species (Kozlowski 1992); and (2) when fast-developing infructescences drain heavily on carbon sources and thereby exceed the carbon-supply capacity of the branchlet they are attached to. Heavy fruiting in masting genera such as Fagus or Quercus has traditionally been regarded as a heavy burden for the carbon budget of the trees (Gäumann 1935; Yamauchi 1996; Hilton & Packham 2003). However, many trees have infructescences with photosynthetically active tissue or leaf-analog organs (e.g. bracts or hypsophylls), the proportion of which within the whole infructescence varies greatly from species to species. Thus, those infructescences with a high proportion of assimilating tissue may not strongly depend on carbon imports, or may even be C-autonomous; at least during the first weeks of their development (Aschan & Pfanz 2003).
The current study is aimed at experimentally exploring the degree of carbon autonomy of fruit-bearing branchlets in mature tree crowns by manipulating the branchlets’ sink–source relationships in situ. The test included the application of defoliation (half and complete defoliation), girdling or the combination of both treatments on fruiting branchlets. Partial or complete defoliation affects carbon fixation in a given branchlet, while girdling, – the removal of a small strip of bark – interrupts the phloem transport of carbohydrates out of and into the terminal branchlet-section beyond the girdle. The translocation of carbohydrates via the xylem is considered of minor significance during most of the vegetation period (Zimmermann 1974; Kozlowski 1992), but to some extent it does occur (Heizmann et al. 2001) and can be quite significant before and during early spring bud break in some species such as Acer saccharum (Larochelle et al. 1998) or Betula pendula (Sauter & Ambrosius 1986).
With few exceptions (e.g. Obeso 1998), previous studies have explored either the effects of defoliation or girdling, but not of both treatments at the same time. Furthermore, most of the previous girdling experiments were conducted in orchard trees aiming at improved fruit quality and yield (e.g. Lavee, Harkad & Ben Tal 1983; Dann, Wildes & Chalmers 1984; Agusti et al. 1998; Mataa, Tominaga & Kozaki 1998). Commercial fruit crops are generally bred for high fruit yield and carbohydrate-rich pulp or oil-rich seeds, which entails that the assimilate-demand for fruit growth is much higher in orchard species than in most forest trees. By using a canopy crane, I was able to manipulate carbon relations of individual shoots during fruit development in the canopy of mature trees of a natural forest in a cross-species comparison.
In addition to the analyses of fruit growth in response to girdling and defoliation, the concentrations of non-structural carbohydrates (NSC) were measured in branchlets and fruits. The quantification of carbon reserves is of special interest since their concentrations reflect the steady state of carbon input and consumption within a plant (Chapin, Schulze & Mooney 1990; Körner 2003) and thus may indicate situations of carbon limitation in fruiting branchlets.
MATERIALS AND METHODS
Study site and sampled trees
The study was performed at the Swiss Canopy Crane (SCC) site near the city of Basel, Switzerland (47°28′N, 7°30′E) (Pepin & Körner 2002). By means of a crane gondola, the upper branches of 32- to 38-m-tall, mature trees could be reached. For the current study the following three deciduous species were investigated: Carpinus betulus L., Tilia platyphyllos SCOP. and Fagus sylvatica L. In C. betulus several (normally 8–14) pairs of seeds (achenes) are growing on one infructescence. Each seed is equipped with one green, approximately 3–5 cm long bract with three lobes (see pictures in Fig. 2). Tilia platyphyllos infructescences are cymes with generally 2–7 globular to ovoid fruits containing one or two seeds. Each cyme bears one, about 5–9 cm long, green hypsophyll. In F. sylvatica 2 (to 4) triangular nuts develop within a thick-walled cupule, which is green initially but gets brown and dry quite early in summer. In the following, the three species will be referred to as Carpinus, Tilia and Fagus. To simplify matters, achenes of Carpinus, fruits of Tilia and nuts of Fagus will be uniformly referred to as fruits.
Girdling and defoliation treatments
Within a tree crown, sun-lit fruit-bearing branchlets were randomly assigned to one of the following six treatments: (1) un-girdled and un-defoliated, namely control; (2) un-girdled and 50% defoliated; (3) un-girdled and 100% defoliated; (4) girdled and un-defoliated; (5) girdled and 50% defoliated; (6) girdled and 100% defoliated (Fig. 1). The combinations of girdling and defoliation allowed assessing different aspects of a branchlet's carbon relation: the development of infructescences on 100% defoliated and un-girdled branchlets depended on the import of stored carbon reserves and current photosynthesis of green fruit tissue (Fig. 1). On branchlets, which were girdled but not defoliated, fruits could be supplied only with assimilates from leaves on the same branchlet or the infructescence itself. Finally, girdling and 100% defoliation left the infructescences with no other carbon source than photosynthesis of the green infructescence tissue (plus the small carbon reserves stored in the branchlet's stem).
Girdling was achieved by carefully removing a small, about 3–5 mm wide, stripe of bark and phloem at the base of the branchlet with a razor blade. Because of the different morphologies of the three species, the girdling was done in different age segments of the branchlets in order to arrive at comparable branchlet sizes (similar stem to leaf mass). The girdling was thus performed at the base of 2-year segments in Carpinus and of 1-year-old-segments in Tilia, whereas in Fagus, shoots of approximately 3–5 years had to be girdled to meet the common size criteria. To minimize post-girdling desiccation, wounds were covered tightly with parafilm. For defoliation, whole leaves were clipped (either all or every second). Each treatment was replicated five times (i.e. 30 treated branchlets per species).
Time of treatment and sampling
The treatments were applied to all three species shortly after pollination and the onset of fruit development on 14 and 15 May 2003. Whole branchlets were harvested close to fruit maturation, prior to fruit fall (1 July 2003 for Tilia and 5 August 2003 for Carpinus and Fagus). Temperatures during the summer of 2003 were exceptionally high (Schär et al. 2004) and therefore fruit development was somewhat ahead of ‘normal’ years. Immediately after harvest, branchlets were separated into: (1) xylem; (2) bark plus phloem; (3) leaves; (4) fruits; and (5) all remaining (vegetative) infructescence tissue (including stalk and bract or cupule). All samples were shock-heated in a microwave immediately after harvest to inactivate enzyme activities (Popp et al. 1996) and oven-dried at 75 °C until weight constancy. The dried samples were weighed and then ground to fine powder and stored dry at 4 °C until chemical analyses.
Biometric and NSC analyses
The dry weight of fruits and the rest of the infructescence were measured separately and the number of infructescences and fruits per infructescence was noted for each branchlet. Immediately after harvest, total leaf area was measured with a photo-planimeter (Leaf area meter, LI 3050 A; Li-Cor, Lincoln, NE, USA). Specific leaf area (SLA) was calculated as cm2 per g dry leaf mass.
The determination of NSC followed a modified method of (Wong 1990) as described in detail in (Hoch, Popp & Körner 2002). In a first step, the quantitatively most important free sugars, namely sucrose, glucose and fructose, were determined photometrically after enzymatic conversions with invertase and phosphoglucose-isomerase. Following an enzymatic degradation of starch to free glucose by a crude fungal amylase (‘Clarase’ from Aspergillus oryzae; Enzyme Solutions Pty Ltd, Crydon South, VIC, Australia), the sum of free sugars plus starch (NSC) was determined in a separate analysis. The concentration of starch was calculated as NSC minus the free sugars. All concentrations are given as percentage of dry biomass. All chemicals and enzymes, except the ‘Clarase’ were purchased from Sigma Diagnostics, St. Louis, MO, USA.
As the forest beneath the crane is species rich (10 tree species), the number of replicate trees per species is restricted. A part of the crane area is additionally used for canopy CO2 enrichment (Pepin & Körner 2002) and was avoided for this study. Thus, for the current tests two individuals of Carpinus and Tilia and three individuals of Fagus were available (all at a safe distance from the CO2-experiment). Manipulation treatments were randomly assigned to branchlets across the available crowns. As only relative changes in response to the treatments and not the absolute differences among the fruiting branchlets were of interest for this study, the relative difference of treated to control branchlets was used for statistical analyses. To check for possible effects of tree individuals, tree identity was added as a nested factor within the treatments to the full factorial analysis of variance (anova) for girdling and defoliation effects. The relative differences were arcsine transformed prior to analyses to meet the requirement of normal distribution and equal variance. All statistical tests were performed with JMP 3.2.2 (SAS Institute, Cary, NC, USA).
Branchlet survival, fruit abscission and leaf traits
Irrespective of the treatment the survival rate was high in all three species. Only in 100% defoliated and girdled branchlets one out of five branchlets died back in Carpinus and Tilia(Table 1). Within the surviving branchlets one 50% defoliated, un-girdled and two 100% defoliated, girdled branchlets of Fagus shed their infructescences before ripening (Table 1). All other branchlets developed fruit-bearing infructescences and kept them until harvest. Although the treatments were applied close to the end of leaf-expansion, the influence of girdling and partial defoliation on the biometry of individual leaves was apparent, although not statistically significant at the 5% level. The specific leaf area (SLA) of leaves on girdled branchlets tended to be smaller than on un-girdled branchlets by the time of harvest (Table 1). In addition, the total leaf area of 0% and 50% defoliated and girdled Carpinus branchlets was 35–40% smaller than that of the corresponding un-girdled branchlets by the time of harvest (Table 1), indicating a negative effect of girdling on the completion of leaf expansion in this species.
Table 1. Mean number of leaves, leaf area (cm2), specific leaf area (SLA, cm2 g−1 DM), infructescence-number, fruit-number per infructescence, branchlets survival-rate and infructescence abscission-rate in response to the girdling and defoliation treatments
Proportion of branchlets surviving until harvest.
b Proportion of surviving branchlets that shed their infructescences before harvest.
In Carpinus, defoliation of un-girdled branchlets did not change either the total infructescence biomass, or the biomass per fruit (Fig. 2). On girdled branchlets however, the biomass decreased with the degree of defoliation. This reduction was similar for the total infructescence mass as for the individual fruit mass, but due to the higher variability of infructescence masses, defoliation and girdling × defoliation effects were significant only for dry mass per fruit (Table 2). In Tilia, by contrast, the biomass of total infructescences and of single fruits decreased significantly on 100% defoliated branchlets, irrespective of whether shoots were girdled or not (Fig. 2), indicating an insufficient ability to import carbon reserves. The biomass of whole Tilia infructescences on girdled, un-defoliated branchlets was slightly higher in comparison with the controls. Due to the similar pattern of un-girdled and girdled branchlets, there was no significant girdling or girdling × defoliation effect but a significant effect of defoliation in Tilia (Table 2). In Fagus, defoliation of un-girdled branchlets led to only insignificant reductions of total infructescence and fruit biomass (Fig. 2). In girdled branchlets, even a 50% reduction of the leaf area did not reduce infructescence drymass. However, completely defoliated, girdled branchlets produced extremely small infructescences and fruits (Fig. 2). The statistics revealed highly significant effects of girdling, defoliation and their interaction for total infructescence masses, whereas for the single fruit mass only defoliation had a significant effect in Fagus (Table 2).
Table 2. Factorial anova for girdling (g) and defoliation (d) effects on total infructescence dry mass, dry mass per fruit, NSC concentrations in branchlet xylem and in fruits
Total infructescence dry mass
Dry mass per fruit
Xylem, NSC conc.
Fruits, NSC conc.
Tree identity was nested within the treatments to test for possible tree-individual effects. Significant (P < 0.05) results are in bold.
g × d
g × d
g × d
To estimate the extent to which fruit production depended on the amount of assimilates supplied by the leaves at the same branchlet, the dry fruit masses were correlated with the total leaf area for un-girdled and girdled branchlets, separately (Fig. 3). In un-girdled branchlets there was no correlation for Carpinus and Fagus but a significant correlation for Tilia (Fig. 3a, c & e), indicating that Carpinus and Fagus compensated the loss of foliation greatly by the import of stored carbon. In all three species, fruit mass and leaf area were correlated better on girdled branchlets. This dependency was especially strong and highly significant for girdled branchlets of Tilia (Fig. 3b, d & f).
An interspecific comparison of the fruit biomasses on 100% defoliated, girdled branchlets relative to the untreated controls mirrored the decreasing size of green infructescence tissue from Carpinus to Tilia to Fagus. Total fruit weight of 100% defoliated and girdled branchlets of Carpinus still reached about 50% of controls, and the biomass per fruit was reduced by only 40% (Fig. 4). In Tilia, total fruit mass and mass per fruit decreased to approximately 35 and 40% of control, respectively. Fagus finally, revealed the most drastic reduction with less than 20% of control for its total fruit mass and about 25% of control for individual fruits (Fig. 4).
The NSC concentrations in branch xylem varied surprisingly little across the treatments, except for the 100% defoliation plus girdling treatment where concentrations decreased by 33% in Carpinus, 35% in Tilia and 56% in Fagus(Fig. 5). There was a significant effect of girdling on xylem NSC in Carpinus and Fagus and a significant girdling × defoliation effect on NSC in Fagus xylem (Table 2). In Tilia xylem NSC changed significantly only with defoliation (Table 2). Tilia xylem of control branchlets had much lower starch concentrations [about 2% dry mass (DM)] than controls in Carpinus (about 10% DM) and Fagus (about 6% DM). NSC reductions in Carpinus and Fagus xylem were mainly due to reduced starch concentrations, whereas the variations of NSC in treated Tilia branchlets resulted largely from decreasing sugar concentrations (Fig. 5).
The NSC concentrations in the branchlet bark (including the phloem) were even more stable than in the xylem. Especially in Carpinus and Tilia concentration changes were insignificant in response to all treatments. Only bark of 100% defoliated and girdled branchlets of Fagus exhibited a significant reduction of NSC concentrations of almost 50% (Fig. 5). In fruits of Carpinus and Tilia, the NSC concentration changes in response to the treatments greatly resembled those of the respective xylems, whereas in Fagus fruits the variation of NSC was high and not significantly different among the treatments (Fig. 5, Table 2).
The unimpaired fruit development on girdled and un-defoliated branchlets from the beginning of the experiment until harvest in all three species, clearly supports the carbon autonomy hypothesis. Hence, as long as the attached leaf area was not reduced, fruit-bearing branchlets of these species did not seem to depend on the import of stored reserves. Even the removal of half of the leaves on girdled branchlets had only insignificant effects on fruit weight or, as in the case of Fagus, none.
Carbon autonomy of fruit-bearing branches was assumed also in several previous studies. Fruit growth on girdled and un-defoliated shoots was not reduced in Ilex aquifolium (Obeso 1998), and 13C labelling in Alnus hirsuta revealed that beside the fruit-bearing shoot only shoots immediately next to it allocated assimilates to the infructescences (Hasegawa et al. 2003). For orchard species, Candolfi-Vasconcelos, Candolfi & Koblet (1994) found no long-distance re-translocation of carbon reserves for fruit maturation in Vitis vinifera under non-stressing conditions. Even higher fruit weights were reported in peach and nectarine varieties after girdling or ringing of fruit-bearing branches (Agusti et al. 1998). Such an ameliorating effect of girdling for fruit development most likely reflects the prevention of carbon export (removal of competitive sinks such as stem and roots) for photo-assimilates. This indicates that, despite the higher carbon demand for fruit development, fruit-bearing branches of some species are net carbon exporters at undisturbed conditions. However, hormonal reactions to the lesion of the phloem, which could stimulate fruit growth may be of importance, too (Cutting & Lyne 1993). In this study the (insignificant) higher fruit weight on girdled, un-defoliated Tilia branchlets may point at a positive girdling effect as well.
In contrast, there are several reports suggesting that fruit formation may rely on the import of stored carbon reserves in some species. Carbon import from non-fruiting crown parts to fruit-bearing shoots during the final stage of fruit ripening was described for peaches (Corelli-Grappadelli, Ravaglia & Asirelli 1996) and apple trees (Hansen & Christensen 1974; Palmer, Cai & Edjamo 1991). By modelling net assimilation and carbon demand of peach shoots, Walcroft et al. (2004) concluded recently that more than half of all investigated fruit-bearing shoots were not carbon self-sufficient. Maybe such high carbon demand in fruit-bearing branches relates to the high total fruit mass and the high fraction of carbohydrate-rich pulp of crop trees, which may not be seen in forest tree species.
Although the trees investigated here are carbon autonomous at the whole branchlet level when undisturbed, fruit-bearing branchlets can import carbon when defoliated. After 100% defoliation, the ability of un-girdled branchlets to import carbon into the developing fruits is high in Carpinus and Fagus; however, it is very limited in Tilia, similar to the behaviour that Stephenson (1980) described for Catalpa speciosa, which aborted 80% of all fruits when 94% of the leaves on the fruiting shoot were removed. Thus, under a situation of reduced foliation (e.g. by herbivores), some species may abandon the branchlet autonomy to enable the successful completion of fruit development, whereas other species do not. The different capabilities of tree species to import carbon into fruit-bearing, defoliated shoots, awaits an explanation.
The species selected for this study varied in their fraction of green infructescence tissue, which in turn affected leaf loss impacts on fruit growth. Thus on girdled, 100% defoliated branchlets the degree of carbon autonomy at the infructescence level decrease from Carpinus, over Tilia to Fagus. The bracts of Carpinus fruits as well as the hypsophylls of Tilia infructescences assimilated enough carbon to permit fruit growth to about half the weight of controls. In contrast, in Fagus, which lacks such ‘leaf-like’ infructescence organs, and the cupules of which become brown early in summer, the girdling plus 100% defoliation treatment resulted in extremely small fruits or complete fruit loss. In addition to photosynthesis of bracts and hypsophylls, the developing, green fruits themselves can assimilate quantitatively significant amounts of carbon (Aschan & Pfanz 2003; Birkhold, Koch & Darnell 1992). Furthermore, there is evidence that fruits generally possess significant concentrations of cytosolic phosphoenolpyruvate carboxylase (PEPC), which might additionally recycle mitochondrial released CO2 (Blanke & Lenz 1989). High net assimilation rates of green fruits have been described for tropical epiphytic species (Zotz, Vollrath & Schmidt 2003), which often grow under high light conditions in the upper tree crowns. Cipollini & Levey (1991) argued that photosynthesis of green fruits could offset respiratory costs, but only under good light conditions.
The concentrations of NSC in branchlet xylem have been analysed to assess possible situations of carbon imbalances induced by the treatments (Chapin et al. 1990). The current study indeed revealed diminished NSC concentrations under those treatments that resulted in decreased fruit growth, but surprisingly, they did not ever lead to complete depleted NSC pools, even under the most severe treatment (i.e. girdling plus 100% defoliation). Although the amount of carbon, which is stored as NSC within the xylem beyond the girdle is very small compared with the amount of carbon needed for fruit production (estimated 1–3% of the final fruit C content), it is nevertheless remarkable that even these reserves were not fully retrieved for fruiting. There may be two explanation for this: (1) critical nutrients other than carbon, such as nitrogen and phosphorus, were also removed from the branchlets by the defoliation treatments, and became limiting; or (2) a certain fraction of the xylem NSC pool cannot be remobilized. This was shown for elder xylem (Ziegler 1964), and, for example, in older stems of heartwood forming trees, the innermost heartwood still contains detectable concentrations of starch, which presumably cannot be remobilized (Magel, Einig & Hampp 2000; Hoch, Richter & Körner 2003). However, one would not expect 50–70% of the entire NSC pool to be retained in such source deprived, young branch sapwood, as in the 100% defoliated, girdled branchlets of this study.
Fruit growth in these mature forest trees did not seem to depend markedly on the remobilization of non-structural carbon pools in other tree organs, at least not as long as the fruit-bearing branchlets are foliated. This even seems to hold for masting Fagus, in which heavy fruiting was often thought to massively stress the tree's carbon reserves (Gäumann 1935). The result from this manipulative test confirms suggestions derived from a previous descriptive study at the same site in which no significant reduction in the NSC pools in masting Fagus sylvatica during the time of fruit ripening had been found (Hoch et al. 2003). Similarly, no negative effect on starch concentrations in non-fruiting shoots and the main trunk were reported for masting Styrax obassia trees (Miyazaki et al. 2002). Perhaps, the ongoing increase of atmospheric CO2 concentrations (more than 30% over the last 150 years) has diminished carbon limitation in naturally grown forest trees (Körner 2003).
This work profited from infrastructure maintained by the Swiss Canopy Crane project (funded by the Swiss National Science Foundation and the Swiss Federal Office of the Environment, BUWAL, to Ch. Körner). I thank Christian Körner for discussing my research and for his helpful comments on the manuscript. I also thank Erwin Armstutz (crane operation) and Carmen Thurnherr (sample preparation) for their help.