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The intensity and frequency of droughts events are projected to increase in future with expected adverse effects for forests. Thus, information on the dynamics of tree water uptake from different soil layers during and after drought is crucial. We applied an in situ water isotopologue monitoring system to determine the oxygen isotope composition in soil and xylem water of European beech with a 2-h resolution together with measurements of soil water content, transpiration and tree water deficit. Using a Bayesian isotope mixing model, we inferred the relative and absolute contribution of water from four different soil layers to tree water use. Beech took up more than 50% of its water from the uppermost 5 cm soil layer at the beginning of the 2018 drought, but then reduced absolute water uptake from the drying topsoil by 84%. The trees were not able to quantitatively compensate for restricted topsoil water availability by additional uptake from deeper soil layers, which is related to the fine root depth distribution. Absolute water uptake from the topsoil was restored to pre-drought levels within 3 wk after rewetting. These uptake patterns help to explain both the drought sensitivity of beech and its high recovery potential after drought release.

The 18 O signature of atmospheric water vapour (δ18 OV ) is known to be transferred via leaf water to assimilates. It remains, however, unclear how the 18 O-signal transfer differs among plant species and growth forms. We performed a 9-hr greenhouse fog experiment (relative humidity ≥ 98%) with 18 O-depleted water vapour (-106.7‰) on 140 plant species of eight different growth forms during daytime. We quantified the 18 O-signal transfer by calculating the mean residence time of O in leaf water (MRTLW ) and sugars (MRTSugars ) and related it to leaf traits and physiological drivers. MRTLW increased with leaf succulence and thickness, varying between 1.4 and 10.8 hr. MRTSugars was shorter in C3 and C4 plants than in crassulacean acid metabolism (CAM) plants and highly variable among species and growth forms; MRTSugars was shortest for grasses and aquatic plants, intermediate for broadleaf trees, shrubs, and herbs, and longest for conifers, epiphytes, and succulents. Sucrose was more sensitive to δ18 OV variations than other assimilates. Our comprehensive study shows that plant species and growth forms vary strongly in their sensitivity to δ18 OV variations, which is important for the interpretation of δ18 O values in plant organic material and compounds and thus for the reconstruction of climatic conditions and plant functional responses.

Aim The aim was to decipher Europe‐wide spatio‐temporal patterns of forest growth dynamics and their associations with carbon isotope fractionation processes inferred from tree rings as modulated by climate warming. Location Europe and North Africa (30‒70° N, 10° W‒35° E). Time period 1901‒2003. Major taxa studied Temperate and Euro‐Siberian trees. Methods We characterize changes in the relationship between tree growth and carbon isotope fractionation over the 20th century using a European network consisting of 20 site chronologies. Using indexed tree‐ring widths (TRWi), we assess shifts in the temporal coherence of radial growth across sites (synchrony) for five forest ecosystems (Atlantic, boreal, cold continental, Mediterranean and temperate). We also examine whether TRWi shows variable coupling with leaf‐level gas exchange, inferred from indexed carbon isotope discrimination of tree‐ring cellulose (Δ13Ci). Results We find spatial autocorrelation for TRWi and Δ13Ci extending over a maximum of 1,000 km among forest stands. However, growth synchrony is not uniform across Europe, but increases along a latitudinal gradient concurrent with decreasing temperature and evapotranspiration. Latitudinal relationships between TRWi and Δ13Ci (changing from negative to positive southwards) point to drought impairing carbon uptake via stomatal regulation for water saving occurring at forests below 60° N in continental Europe. An increase in forest growth synchrony over the 20th century together with increasingly positive relationships between TRWi and Δ13Ci indicate intensifying impacts of drought on tree performance. These effects are noticeable in drought‐prone biomes (Mediterranean, temperate and cold continental). Main conclusions At the turn of this century, convergence in growth synchrony across European forest ecosystems is coupled with coordinated warming‐induced effects of drought on leaf physiology and tree growth spreading northwards. Such a tendency towards exacerbated moisture‐sensitive growth and physiology could override positive effects of enhanced leaf intercellular CO2 concentrations, possibly resulting in Europe‐wide declines of forest carbon gain in the coming decades. Spanish Government, Grant/Award Number: AGL2015‐68274 ‐C3 ‐3‐R; Sixth Framework Programme, Grant/AwardNumber: EVK2‐2001 ‐00237; Seventh Framework Programme, Grant/AwardNumber: COST ‐STSM ‐ECOST ‐STSM ‐FP1304‐140915‐066395 and ERANET‐Mundus program (Grant agreement 20112573)

Rationale The oxygen isotopic composition (here shown as the δ 18O value) of soluble sugars in leaves and phloem tissue holds valuable information about plant functions in response to climatic changes. However, δ 18O analysis of sugars is prone to error, and thoroughly tested methods are lacking. Methods We performed three experiments to test if sample preparation modifies the δ 18O values of sugars. In experiment 1, we tested the effects of oven‐drying versus freeze‐drying, whereas in experiment 2 we focused on the extraction and purification of leaf sugars. In experiment 3, we investigated the exudation and purification of twig phloem sugars as a function of exudation time and different ethylenediaminetetraacetic acid (EDTA) exudation media. Results Freeze‐drying produced more consistent δ 18O values than oven‐drying for sucrose but not for phloem sugars. The extraction and purification of leaf sugars can be performed without a significant modification of their δ 18O values; yet the purified leaf and phloem sugars possessed higher δ 18O values than the fraction of water‐soluble compounds. Moreover, the exudation time significantly modulated the δ 18O values of phloem sugars, which is probably related to changes in the sugar composition. The addition of EDTA did not improve the determination of the δ 18O values of phloem sugars. Conclusions We show that the sample preparation of plant sugars for the reliable determination of δ 18O values requires a strict protocol, which is described in this paper. For phloem sugar, we recommend a maximum exudation time of 1 h to reduce the degradation of sucrose and minimise oxygen isotope exchange reactions between the resulting hexoses and water., Rapid Communications in Mass Spectrometry, 34 (19), ISSN:1097-0231, ISSN:0951-4198

Stable isotope ratios in tree rings have become an important proxy for palaeoclimatology, particularly in temperate regions. Yet temperate forests are often characterized by heterogeneous stand structures, and the effects of stand dynamics on carbon (δ13 C) and oxygen isotope ratios (δ18 O) in tree rings are not well explored. In this study, we investigated long-term trends and offsets in δ18 O and δ13 C of Picea abies and Fagus sylvatica in relation to tree age, size, and distance to the upper canopy at seven temperate sites across Europe. We observed strong positive trends in δ13 C that are best explained by the reconstructed dynamics of individual trees below the upper canopy, highlighting the influence of light attenuation on δ13 C in shade-tolerant species. We also detected positive trends in δ18 O with increasing tree size. However, the observed slopes are less steep and consistent between trees of different ages and thus can be more easily addressed. We recommend restricting the use of δ13 C to years when trees are in a dominant canopy position to infer long-term climate signals in δ13 C when relying on material from shade-tolerant species, such as beech and spruce. For such species, δ18 O should be in principle the superior proxy for climate reconstructions.

Summary Our understanding of how temporal variations of atmospheric water vapour and its isotopic composition (δ18OV) influence water and assimilates in plants remains limited, restricting our ability to use δ18O as a tracer of ecophysiological processes. We exposed oak (Quercus robur) saplings under wet and dry soil moisture conditions to 18O-depleted water vapour (c. − 200‰) at high relative humidity (c. 93%) for 5 h, simulating a fog event. We then traced the step change in δ18OV into water and assimilates (e.g. sucrose, hexoses, quercitol and starch) in the leaf lamina, main veins and twigs over 24 h. The immediate δ18OV effect was highest for δ18O of leaf lamina water, but 40% lower on δ18O of main vein water. To a smaller extent, we also observed changes in δ18O of twig xylem water. Depending on the individual assimilation rate of each plant, the 18O-label was partitioned among different assimilates, with highest changes in δ18O of starch/sucrose and lowest in δ18O of quercitol. Additionally, 18O-label partitioning and allocation towards leaf starch and twig phloem sugars was influenced by the plant water status. Our results have important implications for water isotope heterogeneity in plants and for our understanding of how the δ18O signal is incorporated into biomarkers.

Carbon reserves are important for maintaining tree function during and after stress. Increasing tree mortality driven by drought globally has renewed the interest in how plants regulate allocation of recently fixed C to reserve formation. Three-year-old seedlings of two species (Tilia platyphyllos and Pinus sylvestris) were exposed to two intensities of experimental drought during ~10 weeks, and 13 C pulse labelling was subsequently applied with rewetting. Tracking the 13 C label across different organs and C compounds (soluble sugars, starch, myo-inositol, lipids and cellulose), together with the monitoring of gas exchange and C mass balances over time, allowed for the identification of variations in C allocation priorities and tree C balances that are associated with drought effects and subsequent drought release. The results demonstrate that soluble sugars accumulated in P. sylvestris under drought conditions independently of growth trends; thus, non-structural carbohydrates (NSC) formation cannot be simply considered a passive overflow process in this species. Once drought ceased, C allocation to storage was still prioritized at the expense of growth, which suggested the presence of 'drought memory effects', possibly to ensure future growth and survival. On the contrary, NSC and growth dynamics in T. platyphyllos were consistent with a passive (overflow) view of NSC formation.

Almost no δ(18) O data are available for leaf carbohydrates, leaving a gap in the understanding of the δ(18) O relationship between leaf water and cellulose. We measured δ(18) O values of bulk leaf water (δ(18) OLW ) and individual leaf carbohydrates (e.g. fructose, glucose and sucrose) in grass and tree species and δ(18) O of leaf cellulose in grasses. The grasses were grown under two relative humidity (rH) conditions. Sucrose was generally (18) O-enriched compared with hexoses across all species with an apparent biosynthetic fractionation factor (ebio ) of more than 27‰ relative to δ(18) OLW , which might be explained by isotopic leaf water and sucrose synthesis gradients. δ(18) OLW and δ(18) O values of carbohydrates and cellulose in grasses were strongly related, indicating that the leaf water signal in carbohydrates was transferred to cellulose (ebio = 25.1‰). Interestingly, damping factor pex px , which reflects oxygen isotope exchange with less enriched water during cellulose synthesis, responded to rH conditions if modelled from δ(18) OLW but not if modelled directly from δ(18) O of individual carbohydrates. We conclude that δ(18) OLW is not always a good substitute for δ(18) O of synthesis water due to isotopic leaf water gradients. Thus, compound-specific δ(18) O analyses of individual carbohydrates are helpful to better constrain (post-)photosynthetic isotope fractionation processes in plants.

RATIONALE The oxygen isotope ratio (δ(18)O) of carbohydrates derived from animals, plants, sediments, and soils provides important information about biochemical and physiological processes, past environmental conditions, and geographical origins, which are otherwise not available. Nowadays, δ(18)O analyses are often performed on carbohydrate bulk material, while compound-specific δ(18)O analyses remain challenging and methods for a wide range of individual carbohydrates are rare. METHODS To improve the δ(18)O analysis of individual carbohydrates by gas chromatography/pyrolysis-isotope ratio mass spectrometry (GC/Pyr-IRMS) we developed a new methylation derivatization method. Carbohydrates were fully methylated within 24 h in an easy-to-handle one-pot reaction in acetonitrile, using silver oxide as proton acceptor, methyl iodide as methyl group carrier, and dimethyl sulfide as catalyst. RESULTS The precision of the method ranged between 0.12 and 1.09‰ for the δ(18)O values of various individual carbohydrates of different classes (mono-, di-, and trisaccharides, alditols), with an accuracy of a similar order of magnitude, despite high variation in peak areas. Based on the δ(18)O values of the main isomers, important monosaccharides such as glucose and fructose could also be precisely analyzed for the first time. We tested the method on standard mixtures, honey samples, and leaf carbohydrates extracted from Pinus sylvestris, showing that the method is also applicable to different carbohydrate mixtures. CONCLUSIONS The new methylation method shows unrivalled accuracy and precision for δ(18)O analysis of various individual carbohydrates; it is fast and easy-to-handle, and may therefore find wide-spread application.

Stable oxygen isotope ratios (δ(18) O) in trees from high latitude ecosystems are valuable sources of information for recent and past environmental changes, but the interpretation is hampered by the complex hydrology of forests growing under permafrost conditions, where only a shallow layer of soil thaws in summer. We investigated larch trees (Larix gmelinii) at two sites with contrasting soil conditions in Siberia and determined δ(18) O of water from different soil depths, roots, twigs, and needles as well as δ(18) O of soluble carbohydrates regularly over two growing seasons. A comparison of results from the 2 yrs revealed an unexpected 'inverse' climate-isotope relationship, as dry and warm summer conditions resulted in lower soil and root δ(18) O values. This was due to a stronger uptake of isotopically depleted water pools originating from melted permafrost or previous winter snow. We developed a conceptual framework that considers the dependence of soil water profiles on climatic conditions for explaining δ(18) O in needle water, needle soluble carbohydrates and stem cellulose. The negative feedback of drought conditions on the source isotope value could explain decreasing tree-ring δ(18) O trends in a warming climate and is likely relevant in many ecosystems, where a soil isotope gradient with depth is observed.

RATIONALE We investigated the applicability of tree-ring whole-wood material for δ(18)O and δ(13)C analysis in comparison with the more time- and resource-intensive use of cellulose, by considering possible variability between (i) five different tree species (Fagus sylvatica, Quercus robur, Picea abies, Abies alba, Pseudotsuga menziesii), (ii) two sites that differ in soil moisture, and (iii) climate conditions within a 10-year period. METHODS Stem cores of 30 individual trees (n = 3 trees per each species and site) were sampled from two sites in south Germany (Bavaria), and tree rings within sapwood of the years 2001-2010 were separated. The δ(18)O and δ(13)C values from homogenized tree-ring whole wood and from extracted cellulose were measured by mass spectrometry. Species-specific offsets in isotope values were analyzed and the responses in isotopic signature to climate variability including a single drought event were compared between whole-wood and cellulose. RESULTS A constant offset in δ(18)O values of ca 5‰ between wood and cellulose was observed for most species independent of site conditions, with a significant difference between beech and Douglas-fir, while inter-annual variability was only observed in oak. The offset in δ(13)C values ranged between 1.45 and 1.84‰ across species, sites and years. Both materials generally showed similar strength in responses to temperature, precipitation and soil water availability, particularly for conifers. Resistance to severe drought stress--partly more strongly reflected in the δ(13)C values of cellulose--was lower for conifers than for the deciduous species. CONCLUSIONS Wood material from the sapwood of the studied tree species is as useful as cellulose for studying environmental effects on tree-ring δ(18)O and δ(13)C values at a short-term scale as considered in most ecophysiological studies. The more variable response of oak may require further investigations.

Little is known about the dynamics of concentrations and carbon isotope ratios of individual carbohydrates in leaves in response to climatic and physiological factors. Improved knowledge of the isotopic ratio in sugars will enhance our understanding of the tree ring isotope ratio and will help to decipher environmental conditions in retrospect more reliably. Carbohydrate samples from larch (Larix gmelinii) needles of two sites in the continuous permafrost zone of Siberia with differing growth conditions were analysed with the Compound-Specific Isotope Analysis (CSIA). We compared concentrations and carbon isotope values (δ13C) of sucrose, fructose, glucose and pinitol combined with phenological data. The results for the variability of the needle carbohydrates show high dynamics with distinct seasonal characteristics between and within the studied years with a clear link to the climatic conditions, particularly vapour pressure deficit. Compound-specific differences in δ13C values as a response to climate were detected. The δ13C of pinitol, which contributes up to 50% of total soluble carbohydrates, was almost invariant during the whole growing season. Our study provides the first in-depth characterization of compound-specific needle carbohydrate isotope variability, identifies involved mechanisms and shows the potential of such results for linking tree physiological responses to different climatic conditions.

Regulation of stomatal (gs ) and mesophyll conductance (gm ) is an efficient means for optimizing the relationship between water loss and carbon uptake in plants. We assessed water-use efficiency (WUE)-based drought adaptation strategies with respect to mesophyll conductance of different functional plant groups of the forest understory. Moreover we aimed at assessing the mechanisms of and interactions between water and CO2 conductance in the mesophyll. The facts that an increase in WUE was observed only in the two species that increased gm in response to moderate drought, and that over all five species examined, changes in mesophyll conductance were significantly correlated with the drought-induced change in WUE, proves the importance of gm in optimizing resource use under water restriction. There was no clear correlation of mesophyll CO2 conductance and the tortuosity of water movement in the leaf across the five species in the control and drought treatments. This points either to different main pathways for CO2 and water in the mesophyll either to different regulation of a common pathway.

Will warming lead to an increased use of older soil organic carbon (SOC) by microbial communities, thereby inducing C losses from C-rich alpine soils? We studied soil microbial community composition, activity, and substrate use after 3 and 4 years of soil warming (+4 °C, 2007-2010) at the alpine treeline in Switzerland. The warming experiment was nested in a free air CO2 enrichment experiment using depleted (13)CO2 (δ(13)C = -30‰, 2001-2009). We traced this depleted (13)C label in phospholipid fatty acids (PLFA) of the organic layer (0-5 cm soil depth) and in C mineralized from root-free soils to distinguish substrate ages used by soil microorganisms: fixed before 2001 ('old'), from 2001 to 2009 ('new') or in 2010 ('recent'). Warming induced a sustained stimulation of soil respiration (+38%) without decline in mineralizable SOC. PLFA concentrations did not reveal changes in microbial community composition due to soil warming, but soil microbial metabolic activity was stimulated (+66%). Warming decreased the amount of new and recent C in the fungal biomarker 18:2ω6,9 and the amount of new C mineralized from root-free soils, implying a shift in microbial substrate use toward a greater use of old SOC. This shift in substrate use could indicate an imbalance between C inputs and outputs, which could eventually decrease SOC storage in this alpine ecosystem.

With the aim of evaluating the influence of glacial meltwater signature on tree-ring stable isotopes, we analysed δ18O and δ13C in the tree rings of Larix decidua Mill. specimens growing in the area of an ice-contact lake (Lago Verde, at Miage Glacier, European Alps). Additionally, we analysed δ18O in the glacial meltwater of the lake and of the glacier stream and compared it with the δ18O of precipitation predicted by a spatial model. We found that tree-ring cellulose of trees fed by glacial meltwaters (LVW site) is significantly more depleted in δ18O than at a control site LVM (−0.91‰) fed only by precipitation, thus reflecting the measured higher depletion of glacial meltwaters with respect to local precipitation. δ13C values did not show significant differences in mean values between the two sites but an anomalous correlation with summer temperature was found at the LVW site, probably due to the different responses of trees stomatal conductance. Over the 30-year period of analysis, four years at LVW (1992, 1995, 2003 and 2009) were markedly depleted in δ18O. These years are those when the highest summer temperatures were recorded in the area (the ones during which glacier ablation usually increases and more depleted meltwaters fill the lake), with the exception of 1995 during which high water levels occurred following the year with the second highest summer temperature (1994). Overall, our analysis demonstrates that tree-ring δ18O, driven by the glacial meltwater signature in the lake, can be used for detecting past major glacier runoff events. The proposed approach could also be used for quantifying past glacier runoff and for defining past distribution areas of glacial meltwaters in glacier forefields, thus contributing to past environmental reconstructions and to hazard assessment.

Erica arborea (L) is a widespread Mediterranean species, able to cope with water stress and colonize semiarid environ- ments. The eco-physiological plasticity of this species was evaluated by studying plants growing at two sites with differ- ent soil moistures on the island of Elba (Italy), through dendrochronological, wood-anatomical analyses and stable isotopes measurements. Intra-annual density fluctuations (IADFs) were abundant in tree rings, and were identified as the key parameter to understand site-specific plant responses to water stress. Our findings showed that the formation of IADFs is mainly related to the high temperature, precipita- tion patterns and probably to soil water availability, which differs at the selected study sites. The recorded increase in the 13 C-derived intrinsic water use efficiency at the IADFs level was linked to reduced water loss rather than to increas- ing C assimilation.The variation in vessel size and the differ- ent absolute values of δ 18 O among trees growing at the two study sites underlined possible differences in stomatal control of water loss and possible differences in sources of water uptake. This approach not only helped monitor sea- sonal environmental differences through tree-ring width, but also added valuable information on E. arborea responses to drought and their ecological implications for Mediterranean vegetation dynamics.

Summary How will carbon source–sink relations of 35-yr-old larch trees (Larix decidua) at the alpine treeline respond to changes in atmospheric CO2 and climate? We evaluated the effects of previously elevated CO2 concentrations (9 yr, 580 ppm, ended the previous season) and ongoing soil warming (4 yr, + 4°C). Larch branches were pulse labeled (50 at% 13CO2) in July 2010 to trace fresh assimilates through tissues (buds, needles, bark and wood) and non-structural carbon compounds (NCC; starch, lipids, individual sugars) using compound-specific isotope analysis. Nine years of elevated CO2 did not lead to increased NCC concentrations, nor did soil warming increase NCC transfer velocities. By contrast, we found slower transfer velocities and higher NCC concentrations than reported in the literature for lowland larch. As a result of low dilution with older carbon, sucrose and glucose showed the highest maximum 13C labels, whereas labels were lower for starch, lipids and pinitol. Label residence times in needles were shorter for sucrose and starch (c. 2 d) than for glucose (c. 6 d). Although our treatments showed no persistent effect on larch carbon relations, low temperature at high altitudes clearly induced a limitation of sink activities (growth, respiration, root exudation), expressed in slower carbon transfer and higher NCC concentrations.

RATIONALE Isotope analysis of carbohydrates is important for improved understanding of plant carbon metabolism and plant physiological response to the environment. High-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS) for direct compound-specific δ13C measurements of soluble carbohydrates has recently been developed, but the still challenging sample preparation and the fact that no single method is capable of separating all compounds of interest hinder its wide-spread application. Here we tested in detail a chromatography method in alkaline media. METHODS We examined the most suitable chromatographic conditions for HPLC/IRMS analysis of carbohydrates in aqueous conifer needle extracts using a CarboPac PA20 anion-exchange column with NaOH eluent, paying specific attention to compound yields, carbon isotope fractionation processes and the reproducibility of the method. Furthermore, we adapted and calibrated sample preparation methods for HPLC/IRMS analysis. OnGuard II cartridges were used for sample purification. RESULTS Good peak separation and highly linear and reproducible concentration and δ13C measurements were obtained. The alkaline eluent was observed to induce isomerization of hexoses, detected as reduced yields and 13 C fractionation of the affected compounds. A reproducible pre-purification method providing ~100% yield for the carbohydrate compounds of interest was calibrated. CONCLUSIONS The good level of peak separation obtained in this study is reflected in the good precision and linearity of concentration and δ13C results. The data provided crucial information on the behaviour of sugars in LC analysis with alkaline media. The observations highlight the importance for the application of compound-matched standard solution for the detection and correction of instrumental biases in concentration and δ13C analysis performed under identical chromatographic conditions. The calibrated pre-purification method is well suited for studies with complex matrices that disable the use of a spiked internal standard for the detection of procedural losses. Copyright © 2012 John Wiley & Sons, Ltd.

We investigated whether stand structure modulates the long-term physiological performance and growth of Pinus halepensis Mill. in a semiarid Mediterranean ecosystem. Tree radial growth and carbon and oxygen stable isotope composition of latewood (δ(13)C(LW) and δ(18)O(LW), respectively) from 1967 to 2007 were measured in P. halepensis trees from two sharply contrasting stand types: open woodlands with widely scattered trees versus dense afforested stands. In both stand types, tree radial growth, δ(13)C(LW) and δ(18)O(LW) were strongly correlated with annual rainfall, thus indicating that tree performance in this semiarid environment is largely determined by inter-annual changes in water availability. However, trees in dense afforested stands showed consistently higher δ(18)O(LW) and similar δ(13)C(LW) values compared with those in neighbouring open woodlands, indicating lower stomatal conductance and photosynthesis rates in the former, but little difference in water use efficiency between stand types. Trees in dense afforested stands were more water stressed and showed lower radial growth, overall suggesting greater vulnerability to drought and climate aridification compared with trees in open woodlands. In this semiarid ecosystem, the negative impacts of intense inter-tree competition for water on P. halepensis performance clearly outweigh potential benefits derived from enhanced infiltration and reduced run-off losses in dense afforested stands.

The idea that photosynthesis-weighted tree canopy leaf temperature (T(canδ)) can be resolved through analysis of oxygen isotope composition in tree wood cellulose (δ(18) O(wc)) has led to the observation of boreal-to-subtropical convergence of T(canδ) to c. 20°C. To further assess the validity of the large-scale convergence of T(canδ), we used the isotope approach to perform calculation of T(canδ) for independent δ(18) O(wc) data sets that have broad coverage of climates. For the boreal-to-subtropical data sets, we found that the deviation of T(canδ) from the growing season temperature systemically increases with the decreasing mean annual temperature. Across the whole data sets we calculated a mean T(canδ) of 19.48°C and an SD of 2.05°C, while for the tropical data set, the mean T(canδ) was 26.40 ± 1.03°C, significantly higher than the boreal-to-subtropical mean. Our study thus offers independent isotopic support for the concept that boreal-to-subtropical trees display conserved T(canδ) near 20°C. The isotopic analysis cannot distinguish between the possibility that leaf temperatures are generally elevated above ambient air temperatures in cooler environments and the possibility that leaf temperature equals air temperature, whereas the leaf/air temperature at which photosynthesis occurs has a weighted average of near 20°C in cooler environments. Future work will separate these potential explanations.

This study aimed to evaluate the effects of long-term repeated aerial nitrogen (N) and sulphur (S) misting over tree canopies of a Sitka spruce plantation in Scotland. We combined δ13C and δ18O in tree rings to evaluate the changes in CO2 assimilation (A) and stomatal conductance (gs) and to assess their contribution to variations in the intrinsic water-use efficiency (WUEi, i.e., the A/gs ratio). Measurements of δ15N enabled shifts in the ecosystem N cycling following misting to be assessed. We found that: (i) N applications, with or without S, increased the ratio between A and gs in favour of A, thus supporting a fertilizer effect of added N. (ii) After the treatments ceased, the trees quickly adjusted to the reductions of N deposition, but not to the reduction in S deposition, which had a negative effect on WUEi by reducing A. This indicates that the beneficial role of N deposition may be negated in forests that previously received a high load of acid rain. (iii) δ15N in tree rings reflected the N dynamics caused by canopy retention, with the fingerprint also present in the litter, after the experiment stopped. (iv) Both our results (obtained using canopy applications) and a collection of published data (obtained using soil applications) showed that generally WUEi increased in response to an increase of N applications, with the magnitude of the changes related to soil conditions and the availability of other nutrients. The shifts observed in δ15N in tree rings also suggest that both the quantity of the applied N and its quality, mediated by processes occurring during canopy N retention, are important determinants of the interactions between N and C cycles. Stable isotopes are useful probes to understand these processes and to put the results of short-term experiments into context.

Nitrogen (N) from atmospheric deposition has been shown to be mainly retained in the organic soil layers of temperate forest ecosystems, but the mechanisms and the physico-chemical fractions involved are still poorly defined. We performed a hot-acid hydrolysis on N-15-labelled soil samples collected 1 week, 3 months and 1 year following a single in situ application of either (NO3-)-N-15 or (NH4+)-N-15 in two montane forest ecosystems in Switzerland: Grandvillard (beech forest on a calcareous, well-drained soil, 650 m above sea level) and Alptal (spruce forest on hydromorphic soil, 1200 m above sea level). After (NH4+)-N-15 application, recovery rates in the soil were smaller in Alptal than in Grandvillard through a large rate of absorption by mosses. At both sites, the organic soil layers retained most of the tracers at all three sampling times between 1 week and 1 year. In Grandvillard, the hydrolysable fraction (hydrolysable N : total N) of N-15 was on average 79% and thus similar to the hydrolysable fraction of native N. This similarity is probably because of the rapid incorporation of N into organic molecules, followed by stabilization of the recalcitrant N pool through organo-mineral bonds with soil minerals. In Alptal, the N-15 hydrolysable fraction was greater than that of native N, particularly after (NH4+)-N-15 application (N-15, 84%; native N, 72%). At both sites, N-15 and the fraction of hydrolysable native N remained constant between 1 week and 1 year. This shows that both the recalcitrant and the hydrolysable pools are stable in the mid-to long-term. We present arguments indicating that biological recycling through microbes and plants contributes to the stability of the hydrolysable N fraction.

Current models of leaf water enrichment predict that the differences between isotopic enrichment of water at the site of evaporation (Delta(e)) and mean lamina leaf water enrichment (Delta(L)) depend on transpiration rates (E), modulated by the scaled effective length (L) of water isotope movement in the leaf. However, variations in leaf parameters in response to changing environmental conditions might cause changes in the water path and thus L. We measured the diel course of Delta(L) for (18)O and (2)H in beech seedlings under well-watered and water-limited conditions. We applied evaporative enrichment models of increasing complexity to predict Delta(e) and Delta(L), and estimated L from model fits. Water-limited plants showed moderate drought stress, with lower stomatal conductance, E and stem water potential than the control. Despite having double E, the divergence between Delta(e) and Delta(L) was lower in well-watered than in water-limited plants, and thus, L should have changed to counteract differences in E. Indeed, L was about threefold higher in water-limited plants, regardless of the models used. We conclude that L changes with plant water status far beyond the variations explained by water content and other measured variables, thus limiting the use of current evaporative models under changing environmental conditions.

A common method to estimate the carbon isotopic composition of soil-respired air is to use Keeling plots (delta(13)C versus 1/CO2 concentration). This approach requires the precise determination of both CO2 concentration ([CO2]), usually measured with an infrared gas analyser (IRGA) in the field, and the analysis of delta(13)C by isotope ratio mass spectrometry (IRMS) in the laboratory. We measured [CO2] with an IRGA in the field (n = 637) and simultaneously collected air samples in 12 mL vials for analysis of the 13C values and the [CO2] using a continuous-flow isotope ratio mass spectrometer. In this study we tested if measurements by the IRGA and IRMS yielded the same results for [CO2], and also investigated the effects of different sample vial preparation methods on the [CO2] measurement and the thereby obtained Keeling plot results. Our results show that IRMS measurements of the [CO2] (during the isotope analysis) were lower than when the [CO2] was measured in the field with the IRGA. This is especially evident when the sample vials were not treated in the same way as the standard vials. From the three different vial preparation methods, the one using N2-filled and overpressurised vials resulted in the best agreement between the IRGA and IRMS [CO2] values. There was no effect on the (13)C-values from the different methods. The Keeling plot results confirmed that the overpressurised vials performed best. We conclude that in the cases where the ranges of [CO2] are large (>300 ppm; in our case it ranged between 70 and 1500 ppm) reliable estimation of the [CO2] with small samples using IRMS is possible for Keeling plot application. We also suggest some guidelines for sample handling in order to achieve proper results.

This study tested the hypothesis that purebred Boran (Bos indicus) cows and crossbreds of Boran and Holstein respond differently to long-term changes of feeding level in nutrient partitioning to milk and body fat stores. A total of 27 cows of these two genotypes were subjected either to a low or a high feeding level from their first oestrus as heifers until birth of their third calf. Half of the cows of each genotype were then switched to the other feeding level during the third reproduction cycle. If at all, Boran cows responded to a change in the feeding level almost exclusively by a corresponding change in body weight but not milk yield. Crossbred cows kept continuously on the low feeding level had a lower milk yield than those continuously fed the high level, but lost similar amounts of body weight. In crossbred cows, changing the feeding level from high to low was accompanied by a mobilization of body reserves, whereas a change from low to high level resulted mostly in an increase in milk yield. Certain other genotype differences in metabolic response were obvious from differences in body composition and from the metabolic profile either reflected in blood (particularly insulin-like growth factor I) or in adipose tissue (lipoprotein lipase). Reproductive performance differed between genotypes, with shorter lactations associated with earlier occurrences of the first oestrus in the Boran cows. Generally, feeding history appeared to have at least as much influence on energy partitioning as the actual feeding level. In conclusion, purebred Boran cows seem to react to long-term food fluctuations mainly by mobilizing and restoring body fat reserves, whereas cows crossbred with Holstein tend to spend extra energy preferentially for milk production.

The analysis of 15N in aqueous samples requires the concentration of dissolved nitrogen (N) into a small volume that can be analysed by mass spectrometry. This is conveniently achieved by the NH3 diffusion technique, where NH4+ is captured on small acidified filters enclosed in PTFE. NO3- can be analysed the same way by reducing it to NH4+ with Devarda's alloy. H2SO4 is commonly used for the acidification of the filters. During combustion, however, this acid leads to the production of SO2 and elemental sulphur, which both have detrimental effects on the mass spectrometer. We propose here to replace H2SO4 with citric acid because it is combusted completely to CO2 and H2O in the elemental analyser before entering the mass spectrometer. Citric acid was found to give comparable results in terms of N recovery and 15N values, both for NH4+ and for NO3- samples. Blank samples revealed that N contamination was slightly lower using citric instead of sulphuric acid as acidifier of the glass filters. NH4+ samples first concentrated over cation-exchange columns were strongly acidic and several methods were tested to raise the pH for the subsequent diffusion. These samples gave incomplete N recoveries, but this problem was independent of the acid used on the filters and of the final pH of the sample. Complete recovery was achieved only by increasing the volume of the eluate from the columns. Citric acid can thus generally be recommended instead of H2SO4 for ammonia diffusion.

A computer-controllable mobile system is presented which enables the automatic collection of 33 air samples in the field and the subsequent analysis for delta13C and delta18O stable isotope ratios of a carbon-containing trace gas in the laboratory, e.g. CO2, CO or CH4. The system includes a manifold gas source input for profile sampling and an infrared gas analyzer for in situ CO2 concentration measurements. Measurements of delta13C and delta18O of all 33 samples can run unattended and take less than six hours for CO2. Laboratory tests with three gases (compressed air with different pCO2 and stable isotope compositions) showed a measurement precision of 0.03 per thousand for delta13C and 0.02 per thousand for delta18O of CO2 (standard error (SE), n = 11). A field test of our system, in which 66 air samples were collected within a 24-hour period above grassland, showed a correlation of 0.99 (r2) between the inverse of pCO2 and delta13C of CO2. Storage of samples until analysis is possible for about 1 week; this can be an important factor for sampling in remote areas. A wider range of applications in the field is open with our system, since sampling and analysis of CO and CH4 for stable isotope composition is also possible. Samples of compressed air had a measurement precision (SE, n = 33) of 0.03 per thousand for delta13C and of 0.04 per thousand for delta18O on CO and of 0.07 per thousand for delta13C on CH4. Our system should therefore further facilitate research of trace gases in the context of the carbon cycle in the field, and opens many other possible applications with carbon- and possibly non-carbon-containing trace gases.

The relative contributions of sources of carbon in soils, such as throughfall, litter, roots, microbial decay products and stable organic fractions, to dissolved organic C are controversial. To identify the origin of dissolved organic C, we made use of a 4-year experiment where spruce and beech, growing on an acidic loam and on a calcareous sand, were exposed to increased CO 2 that was depleted in 13 C. We traced the new C inputs from trees into dissolved organic C, into water-extractable organic C, and into several particle-size fractions. In addition, we incubated the labelled soils for 1 year and measured the production of dissolved organic C and CO 2 from new and old soil C. In the soil solutions of the topsoil, the dissolved organic C contained only 5-10% new C from the trees. The δ 13 C values of dissolved organic C resembled those of C pools smaller than 50 μm, which strongly suggests that the major source of dissolved organic C was humified old C. Apparently, throughfall, fresh litter and roots made only minor contributions to dissolved organic C. Water-extractable organic C contained significantly larger fractions of new C than did the natural dissolved organic C (25-30%). The δ 13 C values of the water-extractable organic C were closely correlated with those of sand fractions, which consisted of little decomposed organic carbon. The different origin of dissolved and water-extractable organic C was also reflected in a significantly larger molar UV absorptivity and a smaller natural 13 C abundance of dissolved organic C. This implies that the sampling method strongly influences the characteristics and sources of dissolved organic C. Incubation of soils showed that new soil C was preferentially respired as CO 2 and only a small fraction of new C was leached as dissolved organic C. Our results suggest that dissolved organic C is produced during incomplete decomposition of recalcitrant native C in the soils, whereas easily degradable new components are rapidly consumed by microbes and thus make only a minor contribution to the dissolved C fraction.

○ The temporal variability in δ 18 O of organic matter (δ 18 O org ) in needles and in early- and late-wood of mature spruce trees (Picea abies) was related to changes in climate under field conditions, including soil water δ 18 O (δ 18 O wso ), air humidity, temperature and global radiation. Measured δ 18 O org was compared with values calculated with the Dongmann model extended by a dampening factor (f org ) and a biochemical fractionation parameter (e org ). ○ The short-term variations in δ 18 O org of current-year spruce needles could be explained by the δ 18 O wso in the topsoil layer (r 2 = 0.43). The difference in δ 18 O org between the years was influenced by differences in humidity. A highly significant agreement between measured and modeled δ 18 O org was achieved with f org = 0.2, yet the dampening was not as strong (0.66), if only the δ 18 O org during needle development was considered. The temporal δ 18 O org signal of current-year needles correlated with the corresponding early- or late-wood δ 18 O, although strongly dampened in the tree rings. ○ These results show that the short-term climate is much more strongly reflected in the seasonal oxygen isotope signal of current-year needle organic matter than early- and late-wood. Yet the temporal variations in both tissues, leaf and wood are the same, showing how precisely the isotopic pattern is translated to the tree rings.

• The effects of nitrogen dioxide (NO2) exposure are reported on the physiology, morphology and carbon partitioning of hybrid poplar clone cuttings (Populus ×euramericana) grown under high and low soil nitrogen supply. • Plants were exposed to filtered air or NO2-enriched air (80–135 nl l−1) over 12 wk growth in phytotrons. Stable isotope analysis, combined with CO2 and H2O gas exchange measurements, biomass analysis and morphological development, was used to assess the integrated long-term effects of NO2. • NO2 had no toxic effects. A reduced 15N-isotope ratio indicated incorporation of NO2 while nitrate reductase activity in leaves was stimulated. The two nitrogen sources had differential effects on water use efficiency (WUE): NO2 exposure increased long-term WUE; soil N supply decreased WUE; a result not detectable using growth and short-term gas exchange experiments. Plants benefited from airborne NO2, increasing CO2 assimilation rate and biomass; both N sources increased shoot production at the expense of root growth. NO2 exposure induced leaf formation with reduced stomatal density and increased leaf area. • NO2 exposure might be beneficial although the reduced root: shoot biomass could have a detrimental effect on nutrient balance and drought resistance.

We measured the carbon and oxygen isotopic composition of stem cellulose of Pinus sylvestris, Picea abies, Fagus sylvatica and Fraxinus excelsior. Several sites along a transect of a small valley in Switzerland were selected which differ in soil moisture conditions. At every site, six trees per species were sampled, and a sample representing a mean value for the period from 1940 to 1990 was analysed. For all species, the mean site δ13C and δ18O of stem cellulose are related to the soil moisture availability, whereby higher isotope ratios are found at drier sites. This result is consistent with isotope fractionation models when assuming enhanced stomatal resistance (thus higher δ13C of incorporated carbon) and increased oxygen isotope enrichment in the leaf water (thus higher δ18O) at the dry sites. δ18 O-δ13C plots reveal a linear relationship between the carbon and oxygen isotopes in cellulose. To interpret this relationship we developed an equation which combines the above-mentioned fractionation models. An important new parameter is the degree to which the leaf water enrichment is reflected in the stem cellulose. In the combined model the slope of the δ18O-δ13C plot is related to the sensitivity of the pi/pa of a plant to changing relative humidity.

Drought-induced forest decline, like the Scots pine mortality in inner-Alpine valleys, will gain in importance as the frequency and severity of drought events are expected to increase. To understand how chronic drought affects tree growth and tree-ring delta(13)C values, we studied mature Scots pine in an irrigation experiment in an inner-Alpine valley. Tree growth and isotope analyses were carried out at the annual and seasonal scale. At the seasonal scale, maximum delta(13)C values were measured after the hottest and driest period of the year, and were associated with decreasing growth rates. Inter-annual delta(13)C values in early- and latewood showed a strong correlation with annual climatic conditions and an immediate decrease as a response to irrigation. This indicates a tight coupling between wood formation and the freshly produced assimilates for trees exposed to chronic drought. This rapid appearance of the isotopic signal is a strong indication for an immediate and direct transfer of newly synthesized assimilates for biomass production. The fast appearance and the distinct isotopic signal suggest a low availability of old stored carbohydrates. If this was a sign for C-storage depletion, an increasing mortality could be expected when stressors increase the need for carbohydrate for defence, repair or regeneration.

[1] Tree ring width, density, and ratio of stable isotopes (13C/12C and 18O/16O) in wood and cellulose were determined for larch (Larix sibirica Ledeb.) growing under water deficit conditions in the forest steppe zone in central Siberia (54°24′N, 89°57′E) for the period 1850–2005. Dendroclimatic analysis of the chronologies indicated precipitation to be the most important factor determining indicated parameters. Precipitation of June is significantly correlated with tree ring width and maximum density (r = 0.36 and 0.43, p < 0.05, respectively). Relations of δ13C and δ18O to precipitation are similar, but the most important month is July (r = −0.47, p < 0.05 for δ13C; r = −0.29, p < 0.05 for δ18O). Further, δ18O is positively related to the mean temperatures of July (r = 0.30, p < 0.05). Carbon and oxygen isotope ratios in wood and cellulose showed strong negative trends for the last 100 years. The reason for this decrease could be the observed shift of the beginning of the vegetation period to earlier dates (up to 1 week) in spring and the increased use of precipitation water stored in the soil from October of the previous year, although other explanations cannot be excluded (in particular the physiological effect of increasing atmospheric CO2 also responsible for lower δ13C values). Thus, an earlier start of the vegetation period could lead to tree ring formation during a period with higher water availability, resulting in stronger isotopic fractionation and 13C depletion, also reflected in a higher earlywood to latewood ratio. At the same time, highly 18O depleted water from October precipitation of the previous year is absorbed. The incorporation of this isotopically lighter water during photosynthesis is reflected in the wood and cellulose of tree rings.

summary Ozone (O3) in ambient air was investigated as a possible cause of visible damage to leaves of white clover (Trifolium repens L.) in agricultural areas of Switzerland. Differences in the sensitivity of five commonly mown cultivars were studied, namely Ladino Sacramento, Ladino California, Alban, Sonja and Milkanova. Plants were grown in pots and exposed to O3 using three different experimental systems: (i) exposure to ambient air, (ii) exposure in open-top chambers to filtered air, unfiltered air, or unfiltered air to which O3 was added from 09.00 until 17.00 h during a four-day period, (iii) exposure under controlled environmental condition to filtered air with or without O3 added daily. Occurrence of injury symptoms on field-grown plants was recorded during the summers of 1987 and 1988. Ozone exposure produced visible injury expressed as small necrotic flecks on the tipper leaf surface. Irrespective of the experimental system used, the following order in O3 sensitivity was observed: L. Sacramento ≤ L. California ≪ Alban ≤ Sonja ≤ Milkanova. Injury symptoms were seen when plants were exposed to ambient O3 or to unfiltered air in open-top chambers, but not in charcoal-filtered air. In ambient air, symptoms in the most sensitive cultivars appeared after episodes with 1-h maximum concentrations of above 120-140μg m−3 occurring on several consecutive days. The symptoms of injury could be reproduced in controlled-environment fumigation experiments using concentrations comparable to those observed in ambient air. Plants of the different cultivars grown under controlled environmental conditions did not differ in superoxide dismutase or peroxidase activity, or in the content of fructose, sucrose or glucose. Differences in O3−sensitivity between cultivars were related to differences in stomatal density and to some degree to the length of the stomatal pore. Stomatal densities were related to leaf conductance, transpiration, and rate of water loss by excised leaves. Leaves of Milkanova (resistant) had higher 313values than L. California (sensitive). Compared with filtered air, exposure to unfiltered ambient air or O3–amended air in open-top chambers caused a change in δ13 value in leaves of L. California (sensitive), but not in leaves of Milkanova (resistant). It is concluded that ambient levels of O3 in Switzerland can be sufficient to produce visible leaf injury in sensitive white clover cultivars characterized by a high gas-phase conductance of the leaves, that the isotopic composition of leaf tissue could be used as an indicator of O3 sensitivity in different cultivars, and that ambient levels of O3 can change the isotopic composition of the leaf tissue in sensitive species.