Carbon Allocation into Different Fine-Root Classes of Young Abies alba Trees Is Affected More by Phenology than by Simulated Browsing

Abies alba (European silver fir) was used to investigate possible effects of simulated browsing on C allocation belowground by 13CO2 pulse-labelling at spring, summer or autumn, and by harvesting the trees at the same time point of the labelling or at a later season for biomass and for 13C-allocation into the fine-root system. Before budburst in spring, the leader shoots and 50% of all lateral shoots of half of the investigated 5-year old Abies alba saplings were clipped to simulate browsing. At harvest, different fine-root classes were separated, and starch as an important storage compartment was analysed for concentrations. The phenology had a strong effect on the allocation of the 13C-label from shoots to roots. In spring, shoots did not supply the fine-roots with high amounts of the 13C-label, because the fine-roots contained less than 1% of the applied 13C. In summer and autumn, however, shoots allocated relatively high amounts of the 13C-label to the fine roots. The incorporation of the 13C-label as structural C or as starch into the roots is strongly dependent on the root type and the root diameter. In newly formed fine roots, 3–5% of the applied 13C was incorporated, whereas 1–3% in the ≤0.5 mm root class and 1–1.5% in the >0.5–1.0 mm root class were recorded. Highest 13C-enrichment in the starch was recorded in the newly formed fine roots in autumn. The clipping treatment had a significant positive effect on the amount of allocated 13C-label to the fine roots after the spring labelling, with high relative 13C-contents observed in the ≤0.5 mm and the >0.5–1.0 mm fine-root classes of clipped trees. No effects of the clipping were observed after summer and autumn labelling in the 13C-allocation patterns. Overall, our data imply that the season of C assimilation and, thus, the phenology of trees is the main determinant of the C allocation from shoots to roots and is clearly more important than browsing.


Introduction
Palacio et al. [15], saplings of an approximate height of 30-50 cm height are considered to be one of the most vulnerable stages of a young tree in relation to large herbivore browsing (compare also [6,9]). Thus, in total, 68 five-year-old nursery-grown A. alba saplings (height ±30 cm; provenance Beggingen, 47°47 N, 08°33 E, 650 m a.s.l.) were grown in a nursery soil with a pH of 7.2 and were fertilised once at the age of four years with organic NPK fertiliser (Unikorn II, Hauert, Switzerland; 20 g m -2 ).
In the beginning of April 2008, the tree saplings were excavated with their root systems attached, using a root-cutting machine ('Beetroder', Baertschi Agrartechnik AG, Hüswil, Switzerland). The saplings were then replanted into pots (22.5 cm height, 26 cm in diameter) and assigned to two treatments, 35 plants in a browsing treatment and 33 plants in an untreated control. The browsing treatment was performed at the end of April, just prior to bud burst, when a maximum of browsing by wild ungulates can be assumed [23]. The browsing was carried out according to Häsler et al. [10], with clipping two-thirds of the previous year's leader shoots and one-third of the previous year's shoot growth at every second lateral branch (proportion of buds removed: 56±1.3%), using diagonal pliers to break the plant tissue in a comparable way to ungulate browsing.
All saplings were randomly arranged in four rows of 17 trees and shaded with wooden frames above the trees and shading nets at the sides. Thus, the trees received only about onethird of direct sunlight, simulating conditions as in the forest understory [10]. 13 CO 2 pulse-labelling The 13 CO 2 pulse-labelling was conducted with three randomly assembled groups of A. alba tree individuals. Each group was labelled during one physiologically significant phase, i.e., in spring (early May 2008, subsequent to sprouting; 28 trees), in summer (early July 2008, period of stem growth; 20 trees) and in autumn (end of September 2008, end of the growing season; 12 trees). In total, 60 trees were labelled with 13 CO 2 . In addition to these 60 trees, eight trees (four clipped and four controls) remained unlabelled and were used to calculate the respective 13 C enrichment in the trees (Table 1).
At each labelling date, randomly chosen groups of four A. alba trees were lined up side by side, and the shoots of the four trees (two clipped and two control trees) were covered with one polyethylene bag (volume approx. 300 l, 100 μm thickness) and pulse-labelled with 13 CO 2 . The polyethylene bags were then tightly wrapped at the stems, below the crowns, and sealed with string and adhesive tape. With a syringe, 480 ml of gaseous 13 CO 2 (CIL Cambridge Isotope Laboratories, Switzerland; 280 mg 13 C) were added to each polyethylene bag. This equalled 120 ml 13 CO 2 (70 mg 13 C) for each tree individual. In spring, 7 polyethylen bags with total 28 trees were labelled, in summer 5 bags with 20 trees, and in autumn 3 bags with 12 trees. The addition of the gas was performed following Kagawa et al. [24] and Endrulat et al. [22] to ensure efficient incorporation of the label. After closure of the bags, 2 x 40 ml 13 CO 2 were injected into each bag, followed by another 2 x 40 ml after 15 minutes. The trees were left in the bags for 1 h, before bags were opened for another hour. This procedure was repeated three times within one day. The labelling always started around 11 a.m. and lasted until 4 p.m. (summertime) in the afternoon. Since we aimed that plants were exposed to full sunlight for all three labelling dates, we started close to noon because in autumn fog prevails in the mornings. To reduce water condensation and thus limitation of CO 2 incorporation rate, two electric fans per bag were used to ensure continuous circulation within the bag. The fans were equipped with 80 g silica gel in the front to additionally reduce humidity in the bags. Maximum temperatures at the labelling dates were 21.3°C (May), 23.8°C (July) and 14.5°C (September).

Tree measurements
Before and after the clipping treatment and immediately before each sapling was harvested, non-destructive morphological measurements were carried out for each tree. These measurements included sapling height, length of new leader shoot, number of buds, number of new shoots (terminals and laterals), and number of branches off the main stem. Furthermore, the origin of the new leader was recorded as originating from an apical, interwhorl or secondary whorl bud according to Häsler et al. [10] (S1 Table).

Sampling and harvest
Immediately after each of the last labelling exposures, 10 needles of every labelled tree were randomly sampled, milled and analyzed for the maximum 13 C label in each tree. The maximum 13 C contents in the needles were then used to calculate the relative 13 C contents in the roots. Whole trees were harvested at one to four different time points after the three 13 C-labelling dates: In spring, summer, and autumn 2008, and as well as in spring 2009, i.e., 1, 3, 5, and 12 months after a labelling event (Table 1). Of each harvest, four replicate saplings of both treatments (clipped and control) were harvested, except in the spring 2009, one year after the start (spring+1 yr), when a maximum of three replicates was harvested. At each harvest, two non-labelled control trees were sampled as well.
The whole root systems were carefully washed and partitioned into various root categories using a vernier caliper according to Endrulat et al. [22] to keep track of the spatial distribution of the 13 C-label within the roots; new roots (newly formed white-coloured fine roots), fine roots of various size classes (0.5 mm in diameter, >0.5-1 mm in diameter, >1.0-1.5 in diameter mm, >1.5-2 mm in diameter; brown-coloured), and coarse roots (>2 mm in diameter; brown-coloured).
Branches and needles as well as stems were weighed (fresh weight), frozen in liquid nitrogen, and then stored at -20°C until lyophilisation and biomass (dry weight) determination.

Starch extraction
Starch was extracted from two fine-root classes, from the newly formed fine roots and the fine roots with a diameter 0.5 mm, and in spring, summer, and autumn of 2008. Starch extraction followed a protocol of Smith and Zeeman [25], modified according to Regier et al. [26] and Endrulat et al. [22]. Approximately 60 mg (dry weight; DW) of lyophilized and milled A. alba fine roots were extracted with 80% ethanol, and boiled to gelatinise the starch. Starch was then digested with α-amylase and amyloglucosidase (Roche, Switzerland) in water solution. After digestion, the starch amount was measured as released glucose with a spectrophotometer (Tecan, Switzerland) at 340 nm. For 13 C isotopic measurements, the samples were filtrated with centrifugal Filter Devices (Microcon YM-10; Millipore, Switzerland) in order to take out the enzymes according to Göttlicher et al. [27], and then dried in tin capsules (Säntis Analytical AG, Switzerland) in a vacuum centrifuge (Heto Lab Equipment, Denmark). δ 13 C analyses and isotope calculations Carbon isotope and total C concentration analyses in needles, bulk fine-roots and starch were carried out with an Elemental Analyser (EA-1110, Carlo Erba, Italy; Euro EA 3000, Hekatech, Germany) connected to a continuous flow mass spectrometer (Delta V Advantage, Thermo Fisher Scientific, Germany) at WSL. The measured isotope ratios are expressed in δ-notation in permil units (‰), using Vienna-PeeDee Belemnite (V-PDB) as standard. The precision for 13 C analysis was ±0.2‰.
The amounts of assimilated 13 C were calculated according to Philip and Simard [28] and Keel et al. [29]. δ 13 C (‰) and C (%) of labelled samples and unlabelled controls were used to calculate the 13 C enrichment as described in Endrulat et al. [22]. The needle δ 13 C values immediately after each last pulse-labelling exposure were used as maximum signal of each tree individual according to Keel et al. [29]. The 13 C enrichment (mg) of the needles was set to 100%, and the measured 13 C contents in all fine-root samples were expressed in percentage of the maximum needle signals, and referred to as relative 13 C content (%). This approach enables the comparison of all tree individuals due to the elimination of the signal strength variation caused by variable CO 2 uptake during the labelling.
For 13 C analyses in roots, three fine-root classes were analysed: newly formed fine roots, fine roots of 0.5 mm in diameter, and fine roots of >0.5-1 mm in diameter. For 13 C labelled starch of fine roots, only the newly formed fine roots and the fine roots of 0.5 mm in diameter were analysed.

Statistical analyses
All statistical analyses were performed using STATVIEW 5.0 (SAS Institute Inc., Cary, NC, USA). The effects of the clipping treatment and the various harvesting seasons on plants and root properties were analysed by two-way analyses of variance (ANOVA) with using Fisher's protected least significant difference (PLSD). The data were tested for normal distribution and homogeneity of variance. Starch and relative 13 C content data were log-transformed in order to meet the requested criteria. One data set was not normally distributed, therefore, the non-parametric Mann-Whitney (for two groups) and Kruskal-Wallis tests (for more than two groups) were applied.

Biomass partitioning
The clipping of the trees caused over the whole investigation period a significant decrease of aboveground biomass (-5.8%) as well as of the needles (-13.8%; Tables 2 and 3). Subsequently, this loss resulted in a significant increase of the percentage of belowground biomass (+11.9%; Table 3), as well as in a significant increase of the proportion of biomass in coarse (+8.6%) and fine roots (+16%) ( Tables 2 and 3). The root/shoot ratio of clipped trees remained more or less on a constant level (0.56-0.63), whereas the ratio of control trees at the beginning in spring was low (0.41) and increased constantly until autumn (to 0.60; Table 2).
Both, clipping treatment and harvesting season, influenced the tree parameters significantly, with the one exception of the branches after the clipping treatment ( Table 3). Interactions of the two factors, clipping and harvesting, occurred for the root/shoot ratio, the above-and belowground biomass, the needles, and the fine roots (Table 3). This fact indicates that the effects of clipping upon these tree parameters depend on the harvesting season.
Separating the fine roots into newly formed roots and into different diameter classes (Fig 1), we found that root biomass differed among various fine-root classes in all harvesting seasons with the exception of summer (Table 4). These significant effects were mainly caused by the small biomass of the newly formed fine roots (Fig 1). The clipping treatment caused a significant reduction of fine-root biomass in autumn and spring+1 yr ( Table 4, Fig 1), but not in spring or summer (Table 4).

C in needles and fine-roots
The mean recovery of the 13 C isotope (ratio of incorporated 13 C needles to the amount of 13 C added to one tree) after the pulse-labelling was 71% (±8.2%) for control and 55% (±6.2%) for Table 2. Total biomass (g±SE), root/shoot ratio, and percentage of the various tree components of control (unclipped) and clipped trees for two clipping treatments and four harvesting seasons. Aboveground is the sum of stem, branches, and needles, belowground is the sum of coarse and fine roots. Different letters indicate significant differences among harvesting seasons within one treatment (P<0.05). * P<0.05, ** P<0.01, and *** P<0.001 indicate significant differences between 'Control' and 'Clipped' trees. clipped trees. However, mean δ 13 C values in needles directly after the last labelling exposure varied significantly among trees of the same labelling bag (P0.019 for all bags) and among the three labelling dates (P<0.001). Clipped and unclipped control trees did not differ significantly in their foliar δ 13 C values (P = 0.391), and no significant interaction between treatment and labelling date was found (P = 0.078), indicating similar conditions for all labelling dates and no difference in uptake between the two treatments. Fine roots of the same diameter class of clipped and control trees had variable values in terms of 13 C enrichment, expressed as relative 13 C content (%), and the 13 C enrichment differed strongly among seasons of labelling (Fig 2). Spring labelled trees had the lowest relative 13 C contents (<1%) in the fine roots in comparison to summer and autumn labelled fine roots (Fig 2). Highest relative 13 C contents (3-5%) were observed only in newly formed roots of the summer or the autumn harvests immediately after the 13 CO 2 -labelling (Fig 2). Medium high 13 C contents (1-3%) were recorded in fine roots of 0.5 mm and of >0.5-1.0 mm after the summer and the autumn labellings in all harvesting seasons including spring+1 yr (Fig 2).
Clipping the trees had significantly positive effects on the relative 13 C content of the roots of the 0.5 mm and >0.5-1.0 mm root classes when the trees were labelled in spring (Fig 2), whereas clipping had significantly negative effects on the >0.5-1.0 mm root class when trees were labelled in autumn ( Table 5). The harvesting season had a significantly positive effect on new roots and the 0.5 mm root class after summer labelling, with much higher relative 13 C contents after the summer and autumn labelling ( Table 5, Fig 2).  Table 4. Results of two-way ANOVA testing the effect of the clipping treatment (C) and the various fine-root classes (R; new roots, size classes 0.5 mm, >0.5-1.0 mm, >1.0-1.5 mm, and >1.5-2.0 mm) on fine-root biomass at various harvesting seasons. Significant effects (P<0.05) are given in bold (autumn data were tested with non-parametric tests due to non-normal distribution).

Harvesting season
Clipping (

Starch concentrations and recovered 13 C label in starch
Highest starch concentrations (20-24 mg g -1 ) were found in newly grown fine roots in autumn (S2 Table). The clipping treatment did not significantly influence the starch concentrations in the new roots and the 0.5 mm fine root class, whereas the harvesting season influenced them in both root classes significantly (Table 6). Starch in the fine roots of spring 13 CO 2 -labelled trees was only a little labelled with 13 C, with mean relative 13 C contents in starch below 0.01% (Fig 3). In contrast, plants labelled in summer allocated more label into the roots than those labelled in spring: mean relative 13 C contents of new fine roots were up to 0.06% at the summer harvest (Fig 3). Highest starch 13 C enrichments were found after the autumn labelling, with up to 0.3% in new roots and 0.1% in the 0.5 mm root class (Fig 3). Clipping had no significant effect on the relative 13 C content of fine roots, except for the 0.5 mm root class after spring labelling (Table 6). In contrast, harvesting season had significant effects on 13 C in new roots for the spring labelling as well as for the summer labelling (Table 6).

Discussion
The aim of the present study was to investigate the effects of browsing, simulated through clipping, and of different seasons on the C allocation to the root systems of young Abies alba trees. The simulated browsing reduced the aboveground biomass immediately, since a large proportion of the branches have been cut off. The aboveground biomass was reduced mainly due to the clipped branches and due to the proportion of buds removed, and as a consequence, due to the lower number of new shoots produced in the clipped trees. This reduction is in accordance with other studies, e.g. the study of Hester et al. [9], reporting Pinus sylvestris trees having lost a similar amount of buds (58%) due to browsing, whereas the amount was lower in deciduous trees (43% in Betula pendula and 29% in Sorbus aucuparia, respectively). Nevertheless, we found compensatory growth of aboveground parts, e.g. shoots and needles, one year after clipping, as also reported by others [7,9,30]. More than half of the clipped trees built multiple stem forms, similar to results of Häsler et al. [10]. Clipping also affected belowground growth. Evidence of a short-term root growth due to the clipping of shoots was an up to 46% increased biomass in the fine-roots of the classes >0.5-1.0 mm, >1.0-1.5 mm, and >1.5-2.0 mm at spring and summer harvests. Babst et al. [31] as well found increased export of newly assimilated 11 C (t 1/2 : 20.4 min) to roots in Populus tremuloides following jasmonic acid application (simulating a mechanical damage to leaves after feeding by insects). However, after a longer Table 6. Results of two-way ANOVA of the effect of the clipping treatment (C) and the harvesting seasons (H) (spring, summer, autumn) on the starch concentration and on the relative 13 C content of the starch of two fine-root classes. Significant effects (P<0.05) are given in bold. (-: data not available).

Starch
Labelling time period, as in our study after half a year or longer, simulated browsing usually results in a significantly reduced fine-root biomass of treated trees compared to control trees. Such belowground reductions were found as well in other tree species, such as Quercus spp., Betula pubescens, Fagus sylvatica, and Abies alba, after herbivore damage or defoliation [15,20,21,32,33]. Only a few studies, however, observed no belowground changes [18,34]. Starch concentrations in the fine roots of our study, in contrast to the biomass, did not show any changes due to the clipping treatment. Other studies made the same observation, that a 50% defoliation does not affect the amount of root starch [15,17,18,20,21,35]. Only a severe defoliation (100%), however, is able to diminish root starch significantly as shown by Piper et al. [18] with Nothofagus spp. or Jacquet et al. [17] with Pinus pinaster. Kosola et al. [35] additionally found that starch accumulation after defoliation occurred mainly in August, but not during the other measured seasons (early summer, late autumn). Overall, the above mentioned reports on root biomass and root starch showed that the severity of defoliation and the environmental circumstances (e.g. season of measurement) matter strongly.
Pulse-labelling with 13 CO 2 offers a straightforward method to track recently fixed photoassimilates from the shoots to the roots (e.g. [22,27,36,37]). Concerning our three hypotheses (1-3) postulated earlier, our data supported (1) and (2), but (3) only partly: 1. According to our first hypothesis, the seasons of the 13 C-pulse-labelling and, thus, phenology had a strong effect on the allocation of the 13 C-label from the shoot to the roots. Shoots labelled in spring did not supply the fine-roots with high amounts of the 13 C-label, because the fine-roots had a relatively low 13 C-content (<1%). Labelling in summer and autumn, however, revealed that relatively high amounts of the 13 C-label (1-5%) were allocated to the fine roots. Thus, it can be assumed that photoassimilates from spring mainly remain in the shoots to support shoot growth, whereas photoassimilates from summer and autumn are allocated in larger portions to the fine roots (compare also [38]). Our findings correspond with reports by Ericsson et al. [39] and by Hansen et al. [40], using a 14 CO 2 -label, who observed that spring assimilates were used mainly for aboveground growth, whereas autumn assimilates were allocated mainly to belowground parts as storage. Keel et al. [41] found similarly that 13 C-labels from June were transported to the roots to a lesser amount compared to August. Moreover, Kuptz et al. [42] stated upon their labelling results that 'during spring, only negligible amounts of new photosynthates enter the transfer pool', but 'during early summer, new photosynthates enter the transfer pool, directly supplying growth and being further transported to roots and soils, and during late summer, new photosynthates enter both the transfer and storage pools, supplying both growth and maintenance' [42]. Our isotopic data fully support these findings of Kuptz et al. [42]. Subsequently, highest 13 C-enrichments in the starch in the fine roots were recorded in our study in autumn after autumn labelling.
2. As proposed in our second hypothesis, the incorporation of the 13 C-label into the roots was strongly dependent on the root type and the root diameter. Labelling in summer and autumn showed that relatively high amounts of the 13 C-label (1-5%) were allocated to the fine roots, with 3-5% in newly formed fine roots, 1-3% in the 0.5 mm root class, and 1-1.5% in the >0.5-1.0 mm root class. Comparably, the absolute highest 13 C-values in the starch were measured in the newly formed fine roots after the autumn labelling. Keel et al. [41], recording 13 C-labels in starch likewise, observed highest values in the finest root class.
3. In accordance to our third hypothesis, clipping strongly influenced the amount of allocated 13 C-label as well as the site of incorporation and the form of the incorporated 13 C-label.
Clipping had a significantly positive effects on the 13 C-allocation to fine roots after spring labelling, with significantly higher relative 13 C-contents observed in the 0.5 mm and the >0.5-1.0 mm fine-root classes of clipped trees than those of control trees. Similarly, significantly higher relative 13 C-contents of starch were observed in the 0.5 mm fine-root class of clipped trees after spring labelling. After summer and autumn labelling, however, mainly no effects in the 13 C-allocation to the fine roots were observed, assuming that clipping had not a strong effect on the 13 C-allocation pattern.
Our study showed that clipping resulted in a reduction of the belowground biomass but not in an increase of the starch concentration in the roots. Thus, we conclude that no trade-off between growth and storage occurred. A trade-off would occur only if a C limitation would be present [43][44][45]. A sustained severe defoliation would lead to such a C limitation, with increased storage at the expense of growth enhancing the survival chance [45]. In general, tree growth can be limited by the availability of C within the plant (C limitation) or by the tree's ability to use the available C it has because of e.g. nutrient shortage or environmental conditions (sink limitation) [44]. A growing body of literature suggests that current tree growth is sink limited under most conditions, with much of the evidence deducted from trees' high amounts of nonstructural carbon (NSC) [44]. These conclusions rely on the assumption that C storage is passive and that NSC does not accumulate when growth is C limited. However, storage may be an active process, occurring at the expense of growth [44].
The processes behind the source-to-sink transport within plants are complex. Plants respond to herbivore feeding by increasing the production of the phytohormone jasmonic acid [46]. A systemic jasmonic acid induction of defense responses also occurs from roots to shoots and vice versa, thereby affecting the performance of herbivores. However, jasmonic acid also causes the re-allocation of primary metabolites between roots and shoots, and it controls several developmental processes such as root growth or senescence [46]. The molecular processes behind this source-to-sink transport most likely are phloem sucrose transport and invertases in sink organs, as the latter increase sucrose unloading by converting sucrose to hexoses [47,48].
In conclusion, our data imply that season of C assimilation and, thus, the seasonal phenology of trees is a main driver of C allocation from shoots to roots, and, as seen in our experiment, cleary more decisive than simulated browsing. However, within the fine-root system, the C allocation pattern varied greatly. Fine roots which are highly physiologically active, e.g. newly grown fine roots, received the highest amounts of newly assimilated C, but only after summer and autumn labelling. This indicates that newly formed fine roots are highly active only in the second half of the vegetation period in exploring the soil and storing C.
Supporting Information S1 Table. Number of harvested trees. Number of harvested trees with the varying origins of new leader shoots for two clipping treatments and for four harvesting seasons. Treatments are 'Control' = unclipped trees, and 'Clipped' = clipped trees. (DOCX) S2 Table. Means of starch concentrations. Means of starch concentrations (mg g -1 dry weight, ±SE) in new fine roots and in fine roots of class 0.5 mm for two clipping treatments and for three harvesting seasons. Treatments are 'Control' = unclipped trees, and 'Clipped' = clipped trees. (DOCX)