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- Materials and Methods
Terrestrial carbon (C) fluxes are dominated by two major processes: autotrophic C uptake by primary producers and ecosystem respiration (Gifford, 2003). In Europe, forests are the most abundant terrestrial ecosystems (44% of total land mass; FAO, 2006), fixing up to 3.25 Pg C yr−1 (gross primary productivity (GPP); Schulze et al., 2010). Large amounts of the assimilated C are released back to the atmosphere by ecosystem respiration (Valentini et al., 2000; Knohl et al., 2003; Luyssaert et al., 2010), which is divided into autotrophic plant respiration (on average 53% of GPP) and heterotrophic soil respiration (on average 33% of GPP; Schulze et al., 2010).
Autotrophic respiration is usually measured as the CO2 efflux from plants to the atmosphere and may be partitioned into growth respiration (RG, related to processes such as diameter growth and cell wall thickening) and maintenance respiration (RM, related to processes such as protein and lipid turnover and maintenance of the cellular structure; Amthor, 2000). For adult trees, large seasonal variations in the total CO2 efflux and the contributions of RG and RM have been reported, with RG mainly depending on growth activity and RM on temperature (Stockfors & Linder, 1998; Ceschia et al., 2002; Kuptz et al., 2011). CO2 efflux may also be influenced by respiratory or soil CO2 transported in the xylem (Teskey et al., 2008). Despite the great importance of autotrophic respiration for the global C cycle, knowledge concerning the respiratory C supply system is still limited (Trumbore, 2006; Lehmeier et al., 2008). Large uncertainties remain regarding the origin of C in CO2 efflux, that is, regarding the age, number and magnitude of C sources (Trumbore, 2006; Carbone & Trumbore, 2007; Lehmeier et al., 2008) and the extent to which they contribute to respiration (Lötscher et al., 2004). In particular, only a few studies have addressed the importance of C stores and C turnover for higher plants (Trumbore, 2006).
Throughout the growing season, recent photosynthates are often considered the most important C source for respiration (Scartazza et al., 2004; Wertin & Teskey, 2008; Maier et al., 2009). By contrast, several recent reports on tree, herb and grassland respiration have suggested that respiratory C sources may be of variable age, that is, that respiration may also be largely supplied by storage C (Schnyder et al., 2003; Nogués et al., 2006; Carbone & Trumbore, 2007). Recent photosynthates may supply respiration directly, but they may also pass through storage pools with different turnover rates (Lehmeier et al., 2008). The contribution of these storage pools may vary among species (Keel et al., 2007) and may change with season (Nogués et al., 2006); however, the underlying dynamics are not well understood (Trumbore, 2006). In the case of herbaceous sunflower (Helianthus annuus) and alfalfa (Medicago sativa), Lötscher et al. (2004) suggested that individual C pools may be related to specific respiratory fluxes, that is, that RG is supplied exclusively by recent photosynthates, while RM is supplied by older C. Although previous studies on adult forest trees observed fast intermixing of recent photosynthates with old respiratory substrate pools (Keel et al., 2007; Kodama et al., 2008), it remains obscure whether these short- and long-term C pools are related to RG and RM.
Labeling of canopy air with stable C isotopes may reveal the contribution of recent photosynthates to CO2 efflux (Nogués et al., 2006; Lehmeier et al., 2008) and may also reveal the time lag between photosynthetic C uptake and respiratory C loss (Högberg et al., 2008; Plain et al., 2009). Time lags between C uptake and respiratory loss may serve as an indicator of changes in the source–sink relationships of plant tissues, and C transport rates are assumed to be high during times of active growth (Kozlowski, 1992; Wingate et al., 2010). In the present study, we investigated whether the results obtained by Lötscher et al. (2004) for herbaceous species are valid for adult forest trees, which differ greatly from herbaceous species because of their perennial life-cycle, long C transport pathways and high storage capacity. The present study focused on trunk and coarse-root CO2 efflux, neglecting other large respiratory fluxes, that is, CO2 efflux from leaves, fine roots and the soil. We formulated the hypothesis that the RG of the trunk and coarse roots is directly and exclusively supplied by recent photosynthates. To test this hypothesis, adult individuals of one deciduous and one evergreen tree species, that is, of European beech (Fagus sylvatica) and Norway spruce (Picea abies), respectively, were labeled with stable carbon isotopes three times per year during periods of contrasting contributions of RG to CO2 efflux. In view of our hypothesis, the experimental set-up was designed for labeling mainly rapidly turned-over C pools to detect the direct contribution of current photosynthates to trunk and coarse-root respiration.
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- Materials and Methods
Whole canopies of adult beech and spruce trees were labeled with stable C isotopes during spring, early summer and late summer; that is, at the end of leaf expansion, at maximum rates of trunk growth respiration (RG) and at declining RG, respectively. Distinct seasonal dynamics in the allocation of recent photosynthates to woody tissue CO2 efflux were recorded at two trunk and one coarse-root position, in terms of both the magnitude and the time of label appearance.
Stable C isotope labeling was performed using a free-air approach (isoFACE; for details, see Grams et al., 2011) that applies CO2 directly into the tree canopies via vertically hanging tubes. The Kranzberger Forst study site is characterized by low wind velocities, with above-canopy annual means of < 1.7 m s−1 (Heerdt, 2007). Direct exposure of 13C-depleted CO2 within the canopy resulted in high labeling efficacy even at times with low wind speed (Table 1, Grams et al., 2011). An increase in [CO2] by 100 μmol mol−1 did not affect the stomatal conductance or C translocation rate of trees and only marginally increased photosynthetic discrimination against 13C (Fig. 8, Grams et al., 2011). With the exception of slightly decreased δ13CE of beech coarse roots during the summer experiments, day 0 sampling confirmed no previously applied label remained in respiratory pools of trees at the start of the following labeling experiment.
In both species, CO2 efflux and RG displayed distinct seasonal patterns in trunks and coarse roots (Fig. 3). Both parameters and the contribution of RG to CO2 efflux (Fig. 7) were highest during early summer, suggesting high activity of growth processes such as diameter growth and lignin synthesis (Stockfors & Linder, 1998). In beech, the second highest levels were found in spring (lower trunks) and late summer (coarse roots; Fig. 7), indicating that trunk growth had already started in spring (Maunoury et al., 2007), followed by root growth late in the growing season (Smith & Paul, 1988; Högberg et al., 2010). In spruce, the second highest contributions of RG to CO2 efflux were consistently found during late summer, indicating high activity of growth processes late in the season (Kuptz et al., 2011). Overall, respiration was 3- to 10-fold higher in coarse roots compared with trunks (Fig. 3), probably as a result of higher abundances of respiring cells per volume (Marsden et al., 2008), as the ratio of living cambium : total organ volume increases considerably when organ diameter declines.
As recently discussed in the literature, CO2 dissolved in the xylem sap might influence woody tissue CO2 efflux (Teskey et al., 2008). In our experiments, regression slopes between δ13CE and δ13CP were close to the slope of the 1 : 1 relationship (Fig. 5), indicating that a shift in δ13CP results in the same shift in δ13CE without a perceivable contribution of sap flow dissolved CO2. We therefore conclude that sap flow had no or only a small effect on δ13CE during our experiments, which is consistent with recent findings (Ubierna et al., 2009; Grams et al., 2011; Kuptz et al., 2011). During early summer, the δ13CE of spruce was nearly 2‰ enriched in 13C compared with δ13CP (Fig. 5). A similar trend was seen during the early summer experiment in beech, but this was not as pronounced as in spruce (Fig. 5). This offset may be related to intensive lignin synthesis. As lignin is known to be depleted in 13C compared with most respiratory substrates, CO2 respired during its synthesis might be enriched (Hobbie & Werner, 2004; Bowling et al., 2008). Alternatively, this offset might be explained by a higher dependence on 13C-enriched starch as a respiratory substrate during times of high C demand. This idea is based on diurnal observations of sugar–starch interconversion at the leaf level, whereby starch is accumulated during the light as a result of an excess of sugars, but is metabolized during the night (Bowling et al., 2008; Gessler et al., 2008). Similarly, on the seasonal scale, starch storage pools might be built up or metabolized depending on the excess of, or demand for, phloem sugars (Maunoury et al., 2007; Kuptz et al., 2011).
Distinct seasonal dynamics in fLabel and FLabel-Max were observed in beech, although meteorological conditions were similar between experiments (Fig. 2). In spring, only small amounts of label were found at all three sampling positions, although high rates of RG indicated active growth in lower trunks during this time. The highest amounts of label were recovered in late summer, when RG was already declining (Fig. 7). The observed seasonal patterns may indicate the involvement of two distinct C pools in beech trunks, a transfer and a storage pool. We developed a conceptual two-pool model (Fig. 9) that orientated at the findings by Lötscher et al. (2004). Using this model, we investigated whether their results for herbaceous species may be valid for adult forest trees; that is, whether the transfer and the storage pools might be related to RG and RM, respectively.
Figure 9. Conceptual two-pool model (one transfer and one storage carbon (C) pool) of the substrate supply for lower trunk CO2 efflux of adult beech trees during spring, early summer and late summer. RG and RM are growth and maintenance respiration, respectively. The assumption that RG and RM are supplied by the transfer and storage pools, respectively, was derived from Lötscher et al. (2004). Open arrows denote fluxes of respired CO2, and closed arrows fluxes of respiratory substrate. TBCA, total below-ground carbon allocation. During spring, negligible amounts of new photosynthates enter the transfer pool and both RG and RM are predominately supplied by stored C. During early summer, new photosynthates enter the transfer pool, directly supplying RG and being further transported to roots and soils (i.e. TBCA). During late summer, new photosynthates enter both the transfer and storage pools, supplying both RG and RM. Changes in the box size of the storage pool relate to changes in sapwood sugar concentrations observed by Barbaroux & Breda (2002). The size of open arrows represents the mean rate of lower trunk RG and RM during labeling experiments in beech in comparison to the annual maxima of mean daily CO2 efflux recorded from May 2008 to June 2009.
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In spring, the supply of recent photosynthates to the trunk and coarse-root CO2 efflux of beech was low (Fig. 7). At this time, foliage and shoot development may be the strongest sinks for recent photosynthates (Hansen & Beck, 1994; Dickson et al., 2000) and both RG and RM of trunks appear to be supplied by C stores accumulated during previous years (Fig. 9; Helle & Schleser, 2004; Kagawa et al., 2006; Skomarkova et al., 2006). In early summer, C allocation to beech trunks increased with the demand for growth, but the contribution of RG to CO2 efflux still exceeded the contribution of labeled C (Fig. 7, Mordacq et al., 1986; Hansen & Beck, 1994). During this period, fLabel in trunks declined shortly after the end of label application, suggesting high turnover of recent photosynthates in C pools involved in trunk respiration (Fig. 6). Thus, during this period, labeled C may not have entered the storage pool, which typically has higher half-lives (Lehmeier et al., 2008), and was either respired directly (i.e. to supply growth) or allocated to below-ground sinks (Fig. 7, Hansen & Beck, 1990). By contrast, during late summer, fLabel of trunks reached its highest values and did not decline within a couple of days after the end of the label application. At this time, RM was at least partly supplied by labeled C, as 60% of the CO2 efflux was labeled, while RG accounted for only 0.25% (Fig. 7). The longer label retention time, however, suggested slower label turnover and cycling of labeled C through the storage pool (Figs 6, 9). Thus, our results suggest that, during late summer, labeled C may also be incorporated into the storage pool, as the demand for growth was already declining (Fig. 9).
During late summer, trunks are known to be strong C sinks as a result of the accumulation of C for winter storage and frost hardiness (Hansen & Beck, 1990; Skomarkova et al., 2006). FLabel-Max peaked during late summer in trunks. As a consequence, the size of the storage pool is assumed to be shrinking during spring and early summer and to be refilled during late summer. Accordingly, Barbaroux & Breda (2002) reported a distinct decline in sugar concentration in the youngest 10 tree rings of beech between May and July, followed by an increase later in the season. Assuming the same decline in sapwood sugar concentration (c. 5 mg g−1 dry mass) in our study trees, this would result in an estimated C loss of 3.0 ± 0.3 g m−3 sapwood. This is about half of the C released by RM from May to July, indicating that RM can easily be supplied by stored C throughout spring and early summer.
Overall, during spring, both RG and RM of beech trunks were largely supplied by C stores. Our hypothesis that RG is exclusively supplied by recent photosynthates was rejected for both spring and early summer. Consequently, the findings by Lötscher et al. (2004) regarding the RG of herbaceous species were not applicable to adult beech trees. The finding of Lötscher et al. (2004) that RM was exclusively supplied by storage C could not be invalidated for beech during spring and early summer, but was not supported during late summer, as RM was at least partly supplied by recent photosynthates. However, as our results indicated large amounts of photosynthates refilling the storage pool during late summer, the findings of Lötscher et al. (2004) regarding RM could not be rejected for this time either, and remain possible in adult deciduous beech in all three experiments.
Despite significant seasonal differences in meteorological conditions during the experiments, FLabel-Max in spruce hardly changed throughout the year (Fig. 7). As an evergreen species, spruce displayed less dependence on seasonal accumulation and consumption of storage C, which may be refilled by periods of active photosynthesis during mild winter days (Hansen et al., 1996; Hu et al., 2010; Kuptz et al., 2011). Overall, smaller amounts of label were recovered in spruce compared with beech and fLabel did not decrease after the end of label application. Both observations indicate that recent photosynthates consistently mix with large amounts of old respiratory substrate before they are respired (Nogués et al., 2006). Alternatively, lower label abundances in spruce might be explained by higher maintenance costs of the evergreen foliage compared with that of deciduous beech (Sobrado, 1991). Consequently, lower amounts of recent photosynthates might be transported to trunks and coarse roots.
According to recent results on juvenile trees (W. Ritter, Ecophysiology of Plants, Technische Universität München, pers. comm.), the respiratory supply system of spruce might be represented best by a one-pool model. Gymnosperm trunks usually possess lower amounts of parenchyma cells in trunks than angiosperms (Pallardy & Kozlowski, 2008). In spruce, wood parenchyma sums up to only 4.5–7% of total woody tissue (Schmidt-Vogt, 1986), whereas in beech, ray parenchyma alone comprises c. 20% of total woody tissue (Burgert & Eckstein, 2001). Moreover, in spruce trunks, the total amount of nonstructural carbohydrates strongly decreases from the bark towards the sapwood (Egger et al., 1996; Hoch et al., 2003), whereas in beech trunks, carbohydrates are distributed rather equally throughout the trunk (Barbaroux & Breda, 2002; Hoch et al., 2003). Hence, the storage capacity of spruce trunks may be much smaller than that of beech. This is supported by the kinetics observed in fLabel (Fig. 6), but also by spruce phloem sugar concentrations. In particular, oligosaccharides such as raffinose and stachyose were high (Fig. 5) during spring, while during summer, phloem sugars consisted mainly of disaccharides (sucrose). We assumed that, in spruce, raffinose and stachyose were mainly accumulated as storage C (Fischer & Höll, 1992; Hoch et al., 2003). Such a trend was not observed for beech, as storage C might accumulate within trunks rather than within the phloem. However, we note that FLabel-Max in spruce did not differ between experiments. Thus, the high amount of sugars might not derive from pre-labeling periods only, but substrate might also accumulate in trunks during spring shortly before the onset of growth, as previously observed for starch (Höll, 2000). Nonetheless, it is unlikely that RG in spruce was directly and exclusively supplied by recent photosynthates, as most of the time the contribution of RG to CO2 efflux exceeded FLabel-Max (Fig. 7). Thus, our hypothesis regarding the supply for RG was also rejected for spruce. Conversely, the idea that RM is supplied exclusively by storage C could not be rejected on the basis of our results.
Our interpretation of C allocation of beech and spruce is supported by the transport rates of phloem sugars. Expanding previous “natural abundance approach” studies depending on changes of photosynthetic discrimination by meteorological conditions (Keitel et al., 2003; Werner et al., 2006), we used the isoFACE labeling approach to determine the CTR directly and independently of the weather. Overall, CTRs of 0.06 to 0.62 m h−1 were consistent with previous studies (Keitel et al., 2003; Mencuccini & Hölttä, 2010; Wingate et al., 2010). CTR calculated from time lags between the natural variation in δ13CE and TAir gave similar transport rates to CTR calculated from labeling, supporting the natural abundance approach. C transport in spruce was consistently slower compared with that in beech, which may reflect anatomical differences; for example, sieve cells in gymnosperms vs highly specialized sieve tube systems in angiosperms (Schulz & Thompson, 2001; Wingate et al., 2010). In both species, CTR was highest during early summer (Fig. 8). Phloem sap velocity may be sensitive to temperature (Johnsen et al., 2007), but this cannot explain the seasonal pattern in beech, because TAir was rather similar in the three beech experiments. The seasonal dynamics may rather relate to enhanced C sink strength of the growing plant tissue in early summer (Kozlowski, 1992; Wingate et al., 2010).
Overall, observed seasonal dynamics in the C supply of respiratory processes in the trunk and coarse roots of adult trees suggested marked differences in C turnover between deciduous and evergreen species. The respiratory C supply system of beech was interpreted in relation to two C pools and that of spruce in relation to one C pool, corresponding to their contrasting wood anatomy. C transport rates calculated by the ‘natural abundance’ and ‘labeling’ approaches provided similar results. Our hypothesis that RG was exclusively and directly supplied by recent photosynthates was not supported for either species. We conclude that the contribution of recent photosynthates and C stores to respiration is highly variable in deciduous beech, as recent photosynthates were turned over more rapidly during early summer and more slowly in late summer. Evergreen spruce, by contrast, is deemed to be less dependent on seasonal accumulation and consumption of stored C. Buffering of respiratory C supply by older C, however, was evident throughout the year.
As proposed by Gaudinski et al. (2009), seasonal variations in C turnover and in C stores are important parameters in the modeling of C dynamics in plants. However, such parameters are often not taken into account, as detailed information is lacking. In this respect, the present study expands our overall knowledge of the C turnover of deciduous and evergreen trees, and its findings may be of value in the development of model approaches for autotrophic respiration at the tree and forest ecosystem scales.