Whole-tree seasonal nitrogen uptake and partitioning in adult Fagus sylvatica L. and Picea abies L. [Karst.] trees exposed to elevated ground-level ozone

Environmental Pollution 196 (2015) 511e517

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Whole-tree seasonal nitrogen uptake and partitioning in adult Fagus sylvatica L. and Picea abies L. [Karst.] trees exposed to elevated ground-level ozone €berle b, T. Ro € tzer c, R. Matyssek b R.B. Weigt a, *, K.H. Ha a b c

Laboratory of Atmospheric Chemistry, Paul Scherrer Institute, 5232 Villigen, Switzerland €t München, 85354 Freising, Germany Ecophysiology of Plants, Department Ecology and Ecosystem Management, Technische Universita €t München, 85354 Freising, Germany Forest Yield Science, Department Ecology and Ecosystem Management, Technische Universita

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 January 2014 Received in revised form 14 May 2014 Accepted 23 June 2014 Available online 16 July 2014

The effect of long-term exposure of twice-ambient O3 (2
O3) on whole-tree nitrogen (N) uptake and partitioning of adult beech and spruce was studied in a mixed forest stand, SE-Germany. N uptake as 15N tracer and N pools were calculated using N concentrations and biomass of tree compartments. Wholetree N uptake tended to be lower under 2
O3 in both species compared to trees under ambient O3 (1
O3). Internal partitioning in beech showed significantly higher allocation of new N to roots, with mycorrhizal root tips and fine roots together receiving about 17% of new N (2
O3) versus 7% (1
O3). Conversely, in spruce, N allocation to roots was decreased under 2
O3. These contrasting effects on belowground N partitioning and pool sizes, being largely consistent with the pattern of N concentrations, suggest enhanced N demand and consumption of stored N with higher relevance for tree-internal N cycling in beech than in spruce. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Tropospheric ozone Forest trees European beech Norway spruce Nitrogen partitioning Whole-tree budget

1. Introduction Elevated tropospheric ozone (O3) can affect stomatal conductance in leaves and is known to impair carbon (C) acquisition of trees and subsequent allocation with consequences below-ground (e.g. Matyssek and Sandermann, 2003; Karnosky et al., 2003; Andersen, 2003; Matyssek et al., 2010). This can also affect the uptake and allocation of nutrients, such as nitrogen (N) (Haberer et al., 2007; Samuelson et al., 1996; Luedemann et al., 2005; Zak et al., 2007a). Decreased concentrations of newly acquired N in leaves were found in 60-year-old European beech trees in response to elevated O3, following reduced stomatal conductance and transpiration, and possibly increased below-ground N sink strength (Kitao et al., 2009; Weigt et al., 2012a). However, N concentrations as affected by altered N- and C-allocation do not necessarily reflect N uptake and demand of the whole tree, potentially being biased through resource accumulation or dilution related to growthmediated source-sink balances (e.g. Timmer and Stone, 1978; Timmer and Armstrong, 1987).

* Corresponding author. E-mail address: [email protected] (R.B. Weigt). http://dx.doi.org/10.1016/j.envpol.2014.06.032 0269-7491/© 2014 Elsevier Ltd. All rights reserved.

Tree-internal N cycling that buffers changes in soil nitrogen availability might be affected if storage pools are changed in response to stress. Effects of altered N retranslocation in response to O3, as indicated in mature trees (Samuelson et al., 1996), may become significant in ageing trees which increasingly depend on internal N cycling (e.g. Miller, 1984; Nambiar and Fife, 1991). Changing leaf C/N ratio as a consequence of O3 stress can influence litter decomposition, which in turn may alter N availability (Andersen, 2003). Most studies considering N allocation in trees in response to elevated O3 have been performed with young trees or € viita et al., 2001; Bielenberg seedlings (e.g., Wright et al., 1991; Kyto et al., 2002; Luedemann et al., 2005; Kozovits et al., 2005; Yamaguchi et al., 2010). In young trees, reduced N uptake in beech e at unchanged N allocation e under elevated O3 was coupled with reduced biomass production, while young spruce trees did not show such negative effects (Luedemann et al., 2005; Kozovits et al., 2005). In contrast, to our knowledge, very few comparable studies on adult trees are existing, one focussing on fast-growing pioneer tree species (Zak et al., 2007a), and another on a northern red oak seed orchard (Samuelson et al., 1996), showing partly contrasting effects of O3 on N allocation. Very little is known about long-term effects of O3 exposure on N uptake and allocation of adult trees at established forest stands of late

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successional species. In view of future forest management under current climate change scenarios, knowledge about such effects on nutrient status of different tree species is, however, important. With this study we therefore aim at contributing to filling this gap. A previous study in the same experiment, based on N concentrations, indicated reduced uptake and altered allocation of newly acquired N in adult European beech (Fagus sylvatica L.) in response to elevated O3, but no clear response in Norway spruce (Picea abies L. [Karst.]; Weigt et al., 2012a). In the present study, we quantified concentrations of total and newly acquired nitrogen of above- and belowground tree compartments under experimentally enhanced O3 stress and the biomass of the same trees. This up-scaling allowed for a whole-tree perspective on N pools and partitioning of new N, with outcome for weighting O3 stress-induced changes in N and Callocation in an established forest stand. It was hypothesized that the whole-tree pool sizes and partitioning of new N reflect the pattern of O3-induced changes in concentrations of newly acquired N with stronger effects on beech than on spruce. 2. Materials and methods 2.1. Study site The study was performed on 62-yr-old European beech (Fagus sylvatica L.) and 52-yr-old Norway spruce trees (Picea abies [L.] Karst.) in a mixed forest near Freising, SE-Germany (48 2501200 N, 11390 4200 E; 485 m a.s.l.). The stand was dominated by spruce with scattered groups of beech according to common silvicultural practice in central Europe, and at a stand density of 50.77 m2 ha1 (J. Dieler, pers. comm.). Forest management had been stopped at the research site for the last 30 years allowing only self-thinning (Pretzsch et al., 2010). Long-term (1998e2009) average of annual mean air temperature was 8.3  C and annual precipitation 844 mm (LWF, 2010). The soil is a luvisol derived from loess over tertiary sediments, characterized by silty clay loam, (pH 3 to 4 in the upper soil; Pretzsch et al., 1998). The site nutritional status as monitored by foliar and soil analysis before and since the ozone treatment was at optimum for the study trees regarding central European forest € ttlein et al., 2012; further details in Weigt et al., 2012a; Pretzsch et al., conditions (Go 1998). Throughout the growing seasons (April e September) of 2000 through 2007, five adjacent trees per species within a ground area of 10
10 m were exposed to twiceambient O3 levels (2
O3; 150 nl O3 l1 at maximum) released by a free-air O3 exposure system of a capacity of 2000 m3 canopy volume (‘Kranzberg Ozone Fumigation Experiment’, KROFEX; Nunn et al., 2002; Werner and Fabian, 2002; €berle et al., 2012). A corresponding number of trees of each species under the Ha ambient ozone regime (1
O3) served as control. Within each treatment, the individual tree represented the replication unit. An overview on the general experimental design of this study site is given by Matyssek et al., 2007, 2010. 2.2. Experimental setup

15 ± 1.3 m (spruce) after three years of O3 fumigation. These input parameters did not show any differences between ozone treatments. In cases where the model did not provide separate biomass estimates for plant compartments distinguished in N assessments, the following approaches were employed instead:  Tree crowns: Sun and shade crown organs (buds, foliage, woody axes) were distinguished in terms of N concentrations but not so regarding the modelled biomass. As approximation, mean N concentrations of sun and shadeeexposed organs each were employed, presuming roughly a 50:50 proportion of sun and shade biomass. In spruce, N concentration was measured in current-year (0yr) and one-year-old (1yr) needles and twigs (Weigt et al., 2012a). At the study site, 91% of total needle mass was younger than five-year-old (Nunn et al., 2006), while these age classes typically resemble in dry mass related N concentrations (Schulze et al., 1989). Therefore, the mean across current and one-year-old needles and twigs was presumed representative for calculating tree-level N pools of foliage and woody axes.  Root and mycorrhizal biomass: Total root biomass provided by the model was further distinguished into coarse roots (5 mm diameter), medium-sized roots (2 < 5 mm) and fine roots (<2 mm), according to their biomass proportion assessed through the complete harvest of the 15N-labelled plots (1 m2, each) down to 0.5 m depth. Therefore, the roots of the total plots were dug out, sorted by species, and carefully washed and dried before dry mass was determined. Since the measured root biomasses did not differ between ozone treatments the same biomass proportion of root size classes was applied to all trees independent of O3 regime. Additionally, biomass of mycorrhizal root tips, which were not accounted for by BALANCE, were added to fine root biomass according to the biomass proportion of fine roots and mycorrhizal root tips measured in the upper humus-rich mineral soil horizon (Weigt et al., 2012a). The proportion of mycorrhizal root tips amounted to 19 ± 6 (SD) % of total fine root biomass independent of species and O3 regime.  Stem tissues: The modelled total stem biomass was divided into bark and wood proportions for both O3 regimes to account for stem tissue-specific N concentrations (bark; woodozone, i.e. total stem increment during years 2000 through 2006 with O3 fumigation; and woodpre-ozone, i.e., increment of all years before the O3 fumigation; see Weigt et al., 2012a): For bark biomass, 7.2% (beech) and 9.6% (spruce) of total stem dry mass was presumed according to Dietz (1975); biomass proportion of woodozone and woodpre-ozone was calculated on a stem volume basis, with the stem being regarded as cone-shaped in both species as approximation (including the crown height as vertical prolongation of the stem). Measured tree height in 2006 was used as cone height for calculating the volume of woodozone and tree height in 1999 for woodpre-ozone. Respective diameters were derived from measurements at breast height of each tree, taking into account the mean thickness of living bark (2.6 and 3 mm for beech and spruce, respectively) and extrapolated linearly to the stem basis by the theorem on intersecting lines. Thickness of wood formed during the years of the O3 fumigation (2000 through 2006) amounted to 6.4 and 8.5 mm on average for beech and spruce, respectively, according to dendrological measurements (Forest Yield Science, Dep. Ecology and Ecosystem Management, TU München). Wood density was regarded as being similar between the two distinguished stem wood compartments and O3 regimes as an approximation, although wood density may be reduced by elevated O3 as found in seedlings of loblolly pine and poplar (Edwards et al., 1992; Richet et al., 2011).

Twelve of the twenty study trees were labelled with 15N in order to assess uptake and partitioning of newly acquired nitrogen under 1
O3 and 2
O3. Three 15 trees per species and O3 regime received 10 g of 15NH15 N) dis4 NO3 (98 atom % solved in 70 L of rain water, deployed to 1 m2-plots of soil surface area at a distance of ca. 0.5 m from respective tree stems over seven dates in July 2005, as described in Weigt et al. (2012a). Incorporation of the newly acquired N (i.e. “new N” ¼ Nlabelled) was measured in foliage, buds, twigs, stem tissues and roots from the twelve labelled trees, along with measurements of total N (“new” and “old”) concentrations in all twenty study trees during 2005 and 2006 (see Weigt et al., 2012a). Roots and soil of the plots were sampled for N measurements in October 2006 down to 90 cm depth with detailed distinction between root classes and soil horizons (Weigt et al., 2012a), and completely harvested in March/April 2007 down to 50 cm depth (without distinction between soil horizons), in order to determine the total root biomass of the labelled plots. For assessment of the N partitioning, concentrations of total and labelled N refer to the end of the growing season (late autumn/winter).

Calculation of these compartments resulted in the budgets of biomass and total N (old and new N) (Fig. 1), representing the initial situation as used for assessment of newly acquired (labelled) N. While the whole-tree biomass was similar between O3 treatments and species, biomass partitioning differed between species, particularly with a lower proportion of buds, medium-sized and mycorrhizal fine roots in beech as compared to spruce. In spruce, branches as well as medium-sized and mycorrhizal fine roots showed lower biomass proportion under 2
O3 than under 1
O3 whereas stem biomass was somewhat higher. In contrast, the partitioning of total N differed more strongly between species with more total N being accumulated in the stem tissues and less in the roots of beech as compared to spruce. There were no differences between O3 treatments except for a higher proportion of total N in spruce buds under 2
O3 than under 1
O3. This partitioning of biomass and total N provided the basis for the calculation of the recovery and partitioning of newly acquired N.

2.3. N budget of trees

2.4. Recovery of Nlabelled at the whole-tree level

For obtaining whole-tree N pool sizes, measured concentrations of new and total N in each tree compartment were combined with respective biomass estimates as derived from up-scaling procedures with the tree growth model BALANCE (Pretzsch €tzer et al., 2009). The model calculates growth at 10-day intervals on et al., 2008; Ro the basis of physical and physiological processes that are driven by environmental €tzer influences, and has been validated for a range of different site conditions (Ro et al., 2004, 2005, 2010). Biomass modelling was based on measured breast height diameter of 27 ± 3 (SE) cm (beech) and 33 ± 2.6 cm (spruce), tree height of 26 ± 0.6 m (beech) and 28 ± 1 m (spruce), and crown base of 14 ± 1.5 m (beech) and

The recovery of the labelled nitrogen was assessed at 16 months after application, based on samplings at the end of the growing season. Recovery was calculated for each measured tissue and compartment as: recoveryðcompartmentÞ ¼

Nlabelled ðcompartmentÞ
biomassðcompartmentÞ

Nlabel solution where recovery(compartment) is the proportion of labelled N in a certain tissue or compartment per tree or soil relative to the amount of labelled N applied per each tree; Nlabelled (compartment) is the concentration of labelled N in a tissue or

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513

Tests were performed at a significance level of a ¼ 0.05, using JMP INTRO, Version 5.0.1a (SAS Institute Inc., Cary, NC, USA).

3. Results 3.1. Recovery of labelled N at the whole-tree level Sixteen months after application, approximately 72% of the Nlawas recovered in trees and soil. On average, 23 ± 4(SE) % was recovered in the target trees and 29 ± 3% in the soil across both species and treatments. Recovery in neighbouring trees within a distance of up to 2 m accounted for ca. 14 ± 2% (sum of up to five neighbouring trees per each target tree), showing the 15N-labelled plots to be dominated by the roots of the target trees. Additionally, about 5% of the labelled N was leached out of the soil by runoff (data not shown). Although recovery per whole tree was similar between species, buds and mycorrhizal root tips of beech accumulated significantly higher amounts of Nlabelled than those of spruce (p ¼ 0.0041 and 0.049, respectively; Fig. 2). Under 2
O3, Nlabelled remaining in the soil (down to 90 cm) was slightly higher than under 1
O3 (p ¼ 0.0855), while total recovery in the trees tended to be reduced. Note that although recovery of Nlabelled in soil and roots was based on the labelled plots, complete rooting areas of individual trees were closely reflected, as horizontal transfer of the applied label was small, the Nlabelled at 20 cm distance of plot border being reduced to about 5% of the concentrations within the plot (see Table 1). Similarly to the total recovery per tree, the uptake of new N per unit of whole-tree biomass indicated a (non-significant) reduction under 2
O3 in beech and in spruce (Table 2). When related to root mass (<5 mm), the new N uptake in beech also tended towards reduction under 2
O3.

belled

Fig. 1. Biomass (left) and total nitrogen (right) partitioning of the study trees in winter aspect. Only biomass of beech foliage was added here for ease of comparison between deciduous beech and evergreen spruce. Means ± SE, n ¼ 5 trees.

compartment per unit of dry mass; biomass(compartment) is the total mass of a tissue or compartment per tree, or the soil mass; Nlabel solution is the amount of labelled N applied per each 1 m2 plot (i.e. 3.66 g N plot1). In the case of roots, the biomass refers to the root mass as assessed per 1 m2 plot by harvest down to 0.5 m depth (see above), and including the root mass between 0.5 e 0.9 m depth as extrapolated from soil cores. Regarding soil, mass down to 90 cm depth per 1 m2 plot (and down to 60 cm depth underneath the area surrounding the plots within 30 cm distance from plot border) was extrapolated from soil coring in each plot by 10e20 cm vertical steps with up to 5 cores per layer (Weigt et al., 2012a). Recovery of labelled N in non-labelled neighbouring trees was calculated from average recovery of five measured non-labelled trees (and their respective modelled biomass) situated at distances of 1e2 m from labelled plots, and multiplied by the number of all surrounding trees per each plot within a distance of about 2 m. Understorey vegetation which was present only on some spruce plots (mosses) and adjacent to one of the spruce plots (Dryopteris filix-mas L., Rubus fruticosus L. agg., juvenile Acer pseudoplatanus and Abies alba), was not quantified here as their biomass was negligible.

3.2. Partitioning of new N within trees Although the partitioning of biomass as well as total N differed considerably between beech and spruce trees (cf. Fig. 1), the

2.5. Partitioning of new N (¼Nlabelled) in the whole tree Partitioning of Nlabelled per tree, as the result of the allocation of new N within the tree, was determined on the basis of the recovery in autumn/early winter, with the addition of considering foliage of deciduous beech before leaf fall. The partitioning of Nlabelled in each compartment was calculated as: qNlabelled ðcompartmentÞ ¼

recovery ðcompartmentÞ recovery ðtotal treeÞ

where qNlabelled (compartment) is the proportion of Nlabelled, i.e. new N recovered, in each studied tissue or compartment relative to the total of Nlabelled recovered per tree; recovery(total tree) is the sum of the recovery of Nlabelled of all studied tissues or compartments per tree. Since not all tissues or compartments were analysed for Nlabelled before and after beech leaf fall, some approximations were used: Accounting for Nlabelled partitioning of beech foliage in late summer, the proportions of Nlabelled in branches and stem bark (both measured in winter) were corrected for N retranslocated from leaves before leaf fall as the difference between measured Nlabelled in late summer foliage and shed leaves. The proportion of Nlabelled retranslocated between stem bark and branches was assessed according to the proportion of Nlabelled recovered in stem bark and branches. 2.6. Statistical analysis Data were tested with univariate two-way analysis of variance (ANOVA) with the factors 'species' and 'ozone'. The individual trees or soil plots per tree represented the replication unit with, in general, n ¼ 3 in each treatment (1
O3, 2
O3) and species (beech, spruce). Except for spruce, 1
O3, recovery and partitioning of new N at whole-tree level refers to n ¼ 2 trees, since stem core data of one individual tree was not available here. Least Square Means Student's t-test or Least Square Means Contrasts were applied in the case of multiple or individual comparisons.

Fig. 2. Recovery of labelled N in target tree and soil compartments at 16 months upon 15 N-label application, as means ± SE, with n ¼ 3 trees. Note that recovery in belowground compartments is based on the 1 m2 plots down to 90 cm depth. Recovery assessment in soil includes an additional area surrounding the plots of up to 30 cm distance from plot borderline and down to 60 cm depth.

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Table 1 Biomass proportion of coarse, medium-sized and fine roots assessed from harvest before starting of the new growing season (March 2007) of 1-m2 plots down to 0.5 m soil depth of n ¼ 6 beech and spruce trees each, mean ± SE. Biomass Coarse roots (5 mm) proportion

g m2 Beech Spruce

%

Medium-sized roots (2 < 5 mm)

Fine roots (<2 mm; including mycorrhizal root tips)

g m2

g m2

%

Species

Ozone

Species
ozone

Contrast species

Contrast ozone

Buds

(0.0561) (0.0502)

n.s. n.s.

n.s. n.s.

n.s. n.s.

Foliage

n.s. e

n.s. e

n.s. e

Twigs/branches Stem total Stem, woodpre-ozone Stem, woodozone Stem, bark Roots total Coarse roots Medium roots

n.s. n.s. n.s.

n.s. n.s. (spruce only) n.s. n.s. n.s.

0.0498 (1
O3) (0.0506, 1
O3) n.s. e

n.s. n.s. n.s.

n.s. n.s. n.s.

n.s. n.s. n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s. n.s. n.s. 0.0478 (0.0533)

n.s. n.s. n.s. n.s. n.s.

n.s. n.s. n.s. 0.0214 0.0201

n.s. n.s. n.s. 0.0459 (sp) 0.0461 (sp)

Fine roots

n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

(0.0674) (0.0655) (0.0639) (0.0624)

n.s. n.s. n.s. 0.0089 (1
O3) 0.0091 (1
O3) n.s. n.s. 0.0384 (1
O3) 0.0357 (1
O3)

%

2772 ± 873 82.5 ± 3.7 306 ± 67 9.8 ± 1.9 220 ± 45 7.7 ± 1.9 1843 ± 445 76.7 ± 4.6 282 ± 35 14.7 ± 3.2 165 ± 19 8.5 ± 1.7

Table 2 Uptake of newly acquired N (¼Nlabelled) at the whole-tree level under 1
O3 and 2
O3 (means ± SE). Differences between ozone treatments are not statistically significant.

Uptake of new N per kg tree biomass (g kg1) Uptake of new N per kg root biomass (<5 mm; g kg1) Uptake of new N per kg Ntotal (g kg1)

Table 3 Effects of species and ozone treatments on Nlabelled partitioning within trees according to two-way ANOVA (p-values); significant differences at p < 0.05 are indicated in bold, non-significant trends in parentheses, n.s. ¼ not significant. Within interacting factors, species effects under one O3 treatment or ozone effects of only one species are detected as significant contrasts; b ¼ beech, sp ¼ spruce. Upper and lower values per plant compartment display the situations before and after beech leaf fall, respectively, where different values were obtained.

Beech

Spruce

1
O3 2
O3

0.0017 ± 0.0002 0.0014 ± 0.0003

0.0021 ± 0.0014 0.0017 ± 0.0004

1
O3 2
O3

1.650 ± 0.272 0.884 ± 0.159

1.478 ± 0.893 1.525 ± 0.269

1
O3 2
O3

0.707 ± 0.092 0.623 ± 0.139

0.573 ± 0.379 0.813 ± 0.332

partitioning of new N within the trees was similar in both species, except for buds and medium-sized roots: Buds received slightly more new N at the whole-tree level in beech, whereas mediumsized roots showed less new N as compared to spruce, particularly under 1
O3 (Fig. 3, Table 3). For beech, the situation of both, before and after leaf fall was calculated, demonstrating the shift of foliage N retranslocated to branches and stem tissues. Although concentration of new N in leaves was found to be reduced under 2
O3, particularly in sun-exposed leaves (Weigt et al., 2012a), at the whole-tree level, the new N pool of beech foliage did not clearly differ between the O3 treatments.

Mycorrhizal root tips

(0.0770, b) (0.0723, b) (0.0516, b) 0.0487 (b)

The 2
O3 treatment significantly altered the partitioning of new N in below-ground organs but in opposite directions for the two species: In beech, a higher proportion of the new N remained in mycorrhizal root tips of trees under 2
O3, which was consistent with a similar trend in fine roots (Fig. 3, Table 3). Both compartments together received about 17% of new N under 2
O3 compared to only 7% under 1
O3. In contrast, less new N was allocated to roots under 2
O3 in spruce, particularly regarding medium-sized roots. For beech, these differences in new N allocation between O3 treatments were not related to a change in C allocation within roots, since biomass partitioning did not differ between 1
O3 and 2
O3 trees (cf. Fig. 1). In the case of spruce, a lower biomass of mediumsized and fine roots in 2
O3 trees probably resulted in this reduced allocation as compared to 1
O3, since concentration of newly acquired N was similar between both O3 regimes (Weigt et al., 2012a). Above-ground, the partitioning of new N was not significantly affected by O3 in both species. Overall, beech and spruce showed a contrasting pattern in new N partitioning in response to 2
O3, with a higher proportion of new N remaining in roots of beech whereas in spruce N allocation to roots was decreased. 4. Discussion

Fig. 3. Partitioning of newly acquired (¼labelled) N in trees before beech leaf fall (left) and after leaf fall in winter (middle and right), as means ± SE, with n ¼ 3 trees.

The recovery of the labelled N within the plant-soil system was comparable to other field studies ranging between 56 and 100% in the total tree-soil system and between 2 and 57% in trees (Feigenbaum et al., 1987; Buchmann et al., 1996; Weinbaum and van Kessel, 1998; Schleppi et al., 1999; Gebauer et al., 2000; Nadelhoffer et al., 2004). About 28% of the label was not recovered in the analysed stand compartments, potentially being translocated across roots and soil to outside the labelled plots or lost by denitrification.

R.B. Weigt et al. / Environmental Pollution 196 (2015) 511e517

The higher recovery of Nlabelled in the soil under 2
O3 compared to 1
O3, along with slightly reduced whole-tree Nlabelled recovery in both beech and spruce indicated reduced N uptake under 2
O3. Although only weakly pronounced at the whole-tree level, this trend is in line with lowered Nlabelled concentrations in beech compartments and partly higher concentrations in soil layers of both species under 2
O3 (Weigt et al., 2012a). Decreased N uptake under elevated O3 was also reported from young beech trees, as a result of reduced plant biomass development after two years of 2
O3 fumigation, whereas spruce was less affected (Luedemann et al., 2005). Reduced plant N content under elevated O3 was also observed in other tree species as a result of reduced rates of photosynthesis and plant growth (Zak et al., 2007a; cf. Matyssek et al., 2012). Conversely, N uptake under elevated O3 also depends on the sensitivity of the genotype, so that response can hardly be generalized (Zak et al., 2007b). Differences in the partitioning of new N between O3 treatments were most pronounced in below-ground compartments, with the allocation of new N to roots and mycorrhizae being increased under 2
O3 in beech, and decreased in spruce. This pattern was even more distinct than similar trends observed in concentrations of new N (Weigt et al., 2012a), and resulted from biomass partitioning and the integration over soil depth with varying root N concentrations in both species. While the total N partitioning of beech did not differ between O3 regimes, the increased allocation of the newly acquired N to mycorrhizal fine roots indicate a higher demand in these compartments. This is also supported by lower concentration of total N in mycorrizal root tips and a similar trend for fine roots under 2
O3 (Weigt et al., 2012a). In response to O3 stress, reduced N concentrations in roots and mycorrhizae in beech even with increased allocation of new N suggests increased C investment below-ground (Andersen, 2003; Nikolova et al., 2010). However, we did not observe differences in the partitioning of standing root biomass between the O3 treatments. It is known from other studies that beech may respond to stress, such as by drought or O3, by increasing fine root production and root tip density (Leuschner et al., 2001, 2004; Grebenc and Kraigher, 2007; Nikolova et al., 2009). According to Winwood et al. (2007), O3-induced destruction of leaf cytokinins that typically regulate root growth also appear to be involved in increased root production. Thus, increased root growth dynamics and turnover under 2
O3 may explain the increased allocation of new N in mycorrhizal fine roots of beech. Such stress-induced responses at the root level may be advantageous to ensure nutrient and water access in the long term, however, this could be at the expense of above-ground growth performance. In spruce, the pattern of new N partitioning in roots reflects a similar trend as observed in new N concentration (Weigt et al., 2012a), while the lower root mass of trees under 2
O3 may explain the lower allocation of new N to roots of <5 mm in diameter under 2
O3 compared to 1
O3. However, this difference in modelled root biomass per tree between the ozone treatments was not confirmed by the root biomass assessments of the 1-m2-plots. On the other hand, a similar proportion of total N in roots of both 1
O3 and 2
O3 regimes (cf. Fig. 1) suggests that the lower proportion of new N may be due to impaired N uptake under 2
O3. This is supported by studies on spruce ectomycorrhizal composition at the same site showing lower mycelial biomass and occupation of soil space under 2
O3 (Weigt et al., 2012b; Agerer et al., 2012), while plant available soil nutrients remained higher under 2
O3 than under 1
O3 (unpublished data). In addition, increased C allocation to coarse root respiration (Ritter, pers. comm.) and increased soil respiration under ozone stress (Nikolova et al., 2010) indicated

515

construction and maintenance costs of below-ground compartments to be high in spruce. Increased below-ground N demand in the case of beech and decreased N uptake and supply to above-ground organs may, as a consequence affect N storage pools and internal N cycling. Perturbance in N supply may be particularly important regarding new foliage production in deciduous beech, as buds of the studied trees comprised about 50% of total leaf N demand which was also shown by the proportion of new N (cf. Fig. 3). Therefore, foliage N supply depends to a large extent on the N uptake and bud formation during the previous year (Millard, 1996), while the remaining proportion of at least 50% of leaf N is derived from stem storage tissues (Schulze et al., 2002). Thus, although reduced concentrations of new N in leaves and buds under 2
O3 (Weigt et al., 2012a) were not clearly reflected in whole-tree N partitioning (cf. Fig. 3), the reduced above-ground N supply may decrease N storage pools in the long term. Since buds represented a particularly high sink for newly acquired N in beech trees, receiving about 11% of the new N, compared to only 1% in spruce (cf. Fig. 3), the overall reduced N uptake and allocation to aboveground organs in response to O3 would affect N supply of beech foliage more severely than of spruce. Pronounced impacts of O3 on stomatal conductance (Kitao et al., 2009) and thus, transpiration may partly explain the reduced N supply in beech (Weigt et al., 2012a). Indications of altered retranslocation of N before leaf shedding, e.g. due to accelerated leaf senescence under 2
O3, may further affect N storage pools of these trees (Weigt et al., 2012a). Despite decreased stomatal conductance under 2
O3 in spruce (Nunn et al., 2006), this species showed rather enhanced investment of new N in above-ground organs whereas below-ground investment did not benefit (cf. Fig. 3). Since the applied growth model BALANCE did not include an algorithm depicting O3 impact on tree physiology, but was based on growth status and allometric relationships after three years of O3 fumigation, possible O3-induced changes in tree biomass, particularly of short-lived organs, could only partly detected here. Measured tree height and breast height diameter in 2006 as model input parameters, representing cumulative biomass production, as well as breast height increment of stem woodozone did not differ between the O3 regimes of the studied trees of both species. Nevertheless, Pretzsch et al. (2010) found a reduction in annual stem volume increment of beech under 2
O3 when studying stem shape development along the entire stem height. Furthermore, standing root biomass as assessed in the labelled plots was similar €berle et al., 2012). When between species and O3 regimes (Ha considering that annual stem growth in equivalence to stem volume increment was decreased by 44% in beech (Pretzsch et al. 2010), such an effect might have further decreased above-ground allocation of new N. Hence, the allocation of new N to stem wood built during the years of ozone fumigation (woodozone) would have been reduced from ca. 14% to around 8.5%, supporting our findings of altered allocation pattern under 2
O3. Overall, as hypothesized, new N uptake and partitioning at the whole-tree level in general followed the pattern of new N concentrations in most of the compartments of both species as reported by Weigt et al. (2012a), showing weak effects of O3, but with stronger impact on beech compared to spruce. However, N allocation to the roots was more strongly pronounced when considering pool sizes for both species, while effects on aboveground new N concentrations in beech were minimized at the whole-tree level. Therefore, these moderate effects of O3 indicated both a slightly decreased N uptake and altered allocation of newly acquired N, which probably related to altered carbon pool sizes and production.

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5. Conclusions Assessment of N pools and flux sizes in an established forest stand demonstrated the relevance of O3 impact on N uptake and partitioning and improves our understanding on stand-level N cycling. The distinction between ten different N pools revealed changes in N and C sink dynamics, also of rather short-lived tree organs such as fine roots and mycorrhizal root tips, under the influence of O3 stress. Increased allocation of new N into roots and mycorrhizae in beech indicated higher below-ground N sink strength under 2
O3, with the risk of reduced above-ground tree growth in the long term, whereas, in spruce, ozone effects on new N partitioning were less pronounced. Our findings suggest that even slight ozone effects on N uptake and partitioning of new N in both beech and spruce may drain N storage pools, interfering with internal N cycling. This new evidence provides a basis for numerical modelling of O3 stress on maturing forest trees. Acknowledgements This study was part of the 'SFB607 e Growth and parasite defense e Competition of resources in economic plants from forestry and agronomy', funded by the German Research Foundation DFG. We would like to thank Philip Wipfler and Gerhard Schütze (Forest Yield Science, Department Ecology and Ecosystem Management, TU München) for providing data of breast height diameter measurements of the study trees. Michael Goisser, Joseph Heckmair, Franziska J€ ager, Peter Kuba, Johanna Lebherz, Lena Penzkofer, and Ilse Süß are thanked for their assistance in field and laboratory work. References Agerer, R., Hartmann, A., Pritsch, K., Raidl, S., Schloter, M., Verma, R., Weigt, R., 2012. Plants and their ectomycorrhizosphere: cost and benefit of symbiotic soil organisms. In: Matyssek, R., Schnyder, H., Ernst, D., Munch, J.C., Oßwald, W., Pretzsch, H. (Eds.), Growth and Defence in Plants: Resource Allocation at Multiple Scales, Ecological Studies, vol. 220. Springer, pp. 213e242. Andersen, C.P., 2003. Source-sink balance and carbon allocation below ground in plants exposed to ozone. New Phytol. 157, 213e228. Bielenberg, D.G., Lynch, J.P., Pell, E.J., 2002. Nitrogen dynamics during O3-induced accelerated senescence in hybrid poplar. Plant Cell Environ. 25, 501e512. Buchmann, N., Gebauer, G., Schulze, E.D., 1996. Partitioning of 15N-labeled ammonium and nitrate among soil, litter, below- and above-ground biomass of trees and understorey in a 15-year-old Picea abies plantation. Biogeochemistry 33, 1e23. Dietz, P., 1975. Dichte und Rindengehalt von Industrieholz. Holz als Roh- und Werkstoff 33, 135e141. Edwards, N.T., Edwards, G.L., Kelly, J.M., Taylor Jr., G.E., 1992. Three-year growth responses of Pinus taeda L. to simulated rain chemistry, soil magnesium status, and ozone. Water Air Soil Pollut. 63, 105e118. Feigenbaum, S., Bielorai, H., Erner, Y., Dasberg, S., 1987. The fate of 15N labeled nitrogen applied to mature citrus trees. Plant Soil 97, 179e187. Gebauer, G., Zeller, B., Schmidt, G., May, C., Buchmann, N., Colin-Belgrand, M., Dambrine, E., Marin, F., Schulze, E.D., Bottner, P., 2000. The fate of 15N-labelled nitrogen inputs to coniferous and broadleaf forests. In: Schulze, E.D. (Ed.), Carbon and Nitrogen Cycling in European Forest Ecosystems, Ecological Studies, vol. 142. Springer Verlag, Berlin, Heidelberg, pp. 144e170. Grebenc, T., Kraigher, H., 2007. Changes in the community of ectomycorrhizal fungi and increased fine root number under adult beech trees chronically fumigated with double ambient ozone concentrations. Plant Biol. 9, 279e287. €ttlein, A., Baumgarten, M., Diehler, J., 2012. Site conditions and tree internal Go nutrient partitioning in mature European beech and Norway spruce at the Kranzberger Forst. In: Matyssek, R., Schnyder, H., Ernst, D., Munch, J.C., Oßwald, W., Pretzsch, H. (Eds.), Growth and Defence in Plants: Resource Allocation at Multiple Scales, vol. 220. Ecological Studies, Springer, pp. 193e211. Haberer, K., Grebenc, T., Alexou, M., Geßler, A., Kraigher, H., Rennenberg, H., 2007. Effects of long-term free-air ozone fumigation on d15N and total N in Fagus sylvatica and associated mycorrhizal fungi. Plant Biol. 9, 242e252. €berle, K.H., Weigt, R., Nikolova, P.S., Reiter, I.M., Cermak, J., Wieser, G., Ha € tzer, T., Pretzsch, H., Matyssek, R., 2012. Case study “Kranzberger Blaschke, H., Ro Forst”: growth and defence in European beech (Fagus sylvatica L.) and Norway spruce (Picea abies (L.) Karst.). In: Matyssek, R., Schnyder, H., Ernst, D., Munch, J.C., Oßwald, W., Pretzsch, H. (Eds.), Growth and Defence in Plants:

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