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Stable isotope ratio analysis of tree rings has been widely and successfully applied in recent decades for climatic and environmental reconstructions. These studies were mostly conducted at an annual resolution, considering one measurement per tree ring, often focusing on latewood. However, much more information could be retrieved with high-resolution intra-annual isotope studies, based on the fact that the wood cells and the corresponding organic matter are continuously laid down during the growing season. Such studies are still relatively rare, but have a unique potential for reconstructing seasonal climate variations or short-term changes in physiological plant properties, like water-use efficiency. The reason for this research gap is mostly technical, as on the one hand sub-annual, manual splitting of rings is very tedious, while on the other hand automated laser ablation for high-resolution analyses is not yet well established and available. Here, we give an update on the current status of laser ablation research for analysis of the carbon isotope ratio (δ13C) of wood, describe an easy-to-use laser ablation system, its operation and discuss practical issues related to tree core preparation, including cellulose extraction. The results show that routine analysis with up to 100 laser shot-derived δ13C-values daily and good precision and accuracy (ca. 0.1‰) comparable to conventional combustion in an elemental analyzer are possible. Measurements on resin-extracted wood is recommended as most efficient, but laser ablation is also possible on cellulose extracted wood pieces. Considering the straightforward sample preparation, the technique is therefore ripe for wide-spread application. With this work, we hope to stimulate future progress in the promising field of high-resolution environmental reconstruction using laser ablation.

Comparison of assimilate delta C-13 values estimated from different leaf carbon pools, Picarro measurements, and modelled data demonstrates the potential for misinterpretation arising from bulk organic matter delta C-13 analysis.Sucrose has a unique role in recording environmental and physiological signals during photosynthesis in its carbon isotope composition (delta C-13) and transport of the signal to tree rings. Yet, instead of sucrose, total organic matter (TOM) or water-soluble carbohydrates (WSC) are typically analysed in studies that follow delta C-13 signals within trees. To study how the choice of organic material may bias the interpretation of delta C-13 records, we used mature field-grown Scots pine (Pinus sylvestris) to compare for the first time delta C-13 of different leaf carbon pools with delta C-13 of assimilates estimated by a chamber-Picarro system (delta C-13(A_Picarro)), and a photosynthetic discrimination model (delta C-13(A_model)). Compared with sucrose, the other tested carbon pools, such as TOM and WSC, poorly recorded the seasonal trends or absolute values of delta C-13(A_Picarro) and delta C-13(A_model). Consequently, in comparison with the other carbon pools, sucrose delta C-13 was superior for reconstructing changes in intrinsic water use efficiency (iWUE), agreeing in both absolute values and intra-seasonal variations with iWUE estimated from gas exchange. Thus, deriving iWUE and environmental signals from delta C-13 of bulk organic matter can lead to misinterpretation. Our findings underscore the advantage of using sucrose delta C-13 to understand plant physiological responses in depth.

Incomplete knowledge of carbon (C) allocation dynamics in trees hinders accurate modeling and future predictions of tree growth. We studied C allocation dynamics in a mature Pinus sylvestris L. dominated forest with a novel analytical approach, allowing the first comparison of: (i) magnitude and delta C-13 of shoot, stem and soil CO2 fluxes (A(shoot), R-stem and R-soil), (ii) concentration and delta C-13 of compound-specific and/or bulk non-structural carbohydrates (NSCs) in phloem and roots and (iii) growth of stem and fine roots. Results showed a significant effect of phloem NSC concentrations on tracheid growth, and both variables significantly impacted R-stem. Also, concentrations of root NSCs, especially starch, had a significant effect on fine root growth, although no effect of root NSC concentrations or root growth was detected on R-soil. Time series analysis between delta C-13 of A(shoot) and delta C-13 of R-stem or delta C-13 of R-soil revealed strengthened C allocation to stem or roots under high C demands. Furthermore, we detected a significant correlation between delta C-13 of R-stem and delta C-13 of phloem sucrose and glucose, but not for starch or water-soluble carbohydrates. Our results indicate the need to include C allocation dynamics into tree growth models. We recommend using compound-specific concentration and delta C-13 analysis to reveal C allocation processes that may not be detected by the conventional approach that utilizes bulk organic matter.

Intrinsic water-use efficiency (iWUE), a key index for carbon and water balance, has been widely estimated from tree-ring delta C-13 at annual resolution, but rarely at high-resolution intraseasonal scale. We estimated high-resolution iWUE from laser-ablation delta C-13 analysis of tree-rings (iWUE(iso)) and compared it with iWUE derived from gas exchange (iWUE(gas)) and eddy covariance (iWUE(EC)) data for two Pinus sylvestris forests from 2002 to 2019. By carefully timing iWUE(iso) via modeled tree-ring growth, iWUE(iso) aligned well with iWUE(gas) and iWUE(EC) at intraseasonal scale. However, year-to-year patterns of iWUE(gas), iWUE(iso), and iWUE(EC) were different, possibly due to distinct environmental drivers on iWUE across leaf, tree, and ecosystem scales. We quantified the modification of iWUE(iso) by postphotosynthetic delta C-13 enrichment from leaf sucrose to tree rings and by nonexplicit inclusion of mesophyll and photorespiration terms in photosynthetic discrimination model, which resulted in overestimation of iWUE(iso) by up to 11% and 14%, respectively. We thus extended the application of tree-ring delta C-13 for iWUE estimates to high-resolution intraseasonal scale. The comparison of iWUE(gas), iWUE(iso), and iWUE(EC) provides important insights into physiological acclimation of trees across leaf, tree, and ecosystem scales under climate change and improves the upscaling of ecological models.

Intrinsic water-use efficiency (iWUE) is a key index for evaluating carbon and water balance of terrestrial ecosystems. A common approach to estimate iWUE is via measuring tree-ring stable carbon isotope composition (δ13C), which has been widely applied at annual resolution, but rarely at high-resolution intra-seasonal scale. In this study, we examined the reliability and accuracy of tree-ring δ13C in estimating intra-seasonal iWUE (iWUEiso), with the aid of laser ablation IRMS analysis of tree-rings, by comparing iWUEiso with iWUE derived from leaf gas exchange (iWUEgas) and eddy covariance (iWUEEC) data for two Pinus sylvestris L. dominated boreal forests from 2002 to 2019. By carefully timing iWUEiso via modelled yearly tree-ring growth, intra-seasonal variability of iWUEiso aligned well with that of iWUEgas and iWUEEC. However, absolute values of iWUEiso were apparently higher than that of iWUEgas and iWUEEC, which could be ascribed to post-photosynthetic 13C-enrichment from leaf assimilates to tree-rings and to nonexplicit inclusion of mesophyll and photorespiration terms in iWUEiso estimation. These two factors overestimated iWUEiso by up to 11% and 14%, respectively. Our study demonstrates that tree-ring δ13C data can be used to trace intra-seasonal variability of iWUE, and thus provides a valuable tool for detecting short-term dynamics in carbon and water trade for areas and periods with no instrumental data. We encourage more comparisons between iWUEgas, iWUEiso and iWUEEC, which can deepen our understanding of how trees physiologically adapt to climate change across leaf, tree and ecosystem levels.

Stable isotope ratio analysis of tree rings has been widely and successfully applied in recent decades for climatic and environmental reconstructions. These studies were mostly conducted at an annual resolution, considering one measurement per tree ring, often focusing on latewood. However, much more information could be retrieved with high-resolution intra-annual isotope studies, based on the fact that the wood cells and the corresponding organic matter are continuously laid down during the growing season. Such studies are still relatively rare, but have a unique potential for reconstructing seasonal climate variations or short-term changes in physiological plant properties, like water-use efficiency. The reason for this research gap is mostly technical, as on the one hand sub-annual, manual splitting of rings is very tedious, while on the other hand automated laser ablation for high-resolution analyses is not yet well established and available. Here, we give an update on the current status of laser ablation research for analysis of the carbon isotope ratio (δ13C) of wood, describe an easy-to-use laser ablation system, its operation and discuss practical issues related to tree core preparation, including cellulose extraction. The results show that routine analysis with up to 100 laser shot-derived δ13C-values daily and good precision and accuracy (ca. 0.1‰) comparable to conventional combustion in an elemental analyzer are possible. Measurements on resin-extracted wood is recommended as most efficient, but laser ablation is also possible on cellulose extracted wood pieces. Considering the straightforward sample preparation, the technique is therefore ripe for wide-spread application. With this work, we hope to stimulate future progress in the promising field of high-resolution environmental reconstruction using laser ablation.

Intrinsic water-use efficiency (iWUE), a key index for carbon and water balance, has been widely estimated from tree-ring δ

The tree-ring stable C, O and H isotope compositions have proven valuable for examining past changes in the environment and predicting forest responses to environmental change. However, we have not yet recovered the full potential of this archive, partly due to a lack understanding of fractionation processes resulting from methodological constraints. With better understanding of the biochemical and tree physiological processes that lead to differences between the isotopic compositions of primary photosynthates and the isotopic compositions of substrates deposited in stem xylem, more reliable and accurate reconstructions could be obtained. Furthermore, by extending isotopic analysis of tree-ring cellulose to intra-molecular level, more information could be obtained on changing climate, tree metabolism or ecophysiology. This chapter presents newer methods in isotope research that have become available or show high future potential for fully utilising the wealth of information available in tree-rings. These include compound-specific analysis of sugars and cyclitols, high spatial resolution analysis of tree rings with UV-laser, and position-specific isotope analysis of cellulose. The aim is to provide the reader with understanding of the advantages and of the current challenges connected with the use of these methods for stable isotope tree-ring research.

We explore needle sugar isotopic compositions (δ

We investigated the interaction between fungal communities of soil and dead wood substrates. For this, we applied molecular species identification and stable isotope tracking to both soil and decaying wood in an unmanaged boreal Norway spruce-dominated stand. Altogether, we recorded 1990 operational taxonomic units, out of which more than 600 were shared by both substrates and 589 were found to exclusively inhabit wood. On average the soil was more species-rich than the decaying wood, but the species richness in dead wood increased monotonically along the decay gradient, reaching the same species richness and community composition as soil in the late stages. Decaying logs at all decay stages locally influenced the fungal communities from soil, some fungal species occurring in soil only under decaying wood. Stable isotope analyses suggest that mycorrhizal species colonising dead wood in the late decay stages actively transfer nitrogen and carbon between soil and host plants. Most importantly, Piloderma sphaerosporum and Tylospora sp. mycorrhizal species were highly abundant in decayed wood. Soil- and wood-inhabiting fungal communities interact at all decay phases of wood that has important implications in fungal community dynamics and thus nutrient transportation.

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.

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.

Although the instrumental coupling of gas chromatography pyrolysis isotope ratio mass spectrometry (GC Py IRMS) for compound specific O 18 analysis has been commercially available for more than a decade this method has been hardly applied so far. Here we present the first GC Py IRMS O 18 results for trimethylsilyl derivatives of plant sap relevant sugars and a polyalcohol (glucose fructose sucrose raffinose and pinitol). Particularly we focus on sucrose which is assimilated in leaves and which is the most important transport sugar in plants and hence of utmost relevance in plant physiology and paleoclimate studies. Replication measurements of sucrose standards and concentration series indicate that the GC Py IRMS O 18 measurements are not stable over time and that they are amount (area) dependent. We therefore suggest running sample batch replication measurements in alternation with standard concentration series of reference material. This allows for carrying out (i) a drift correction (ii) a calibration against reference material and (iii) an amount (area) correction. Tests with O 18 enriched water do not provide any evidence for oxygen isotope exchange reactions affecting sucrose and raffinose. We present the first application of GC Py IRMS O 18 analysis for sucrose from needle extract (soluble carbohydrate) samples. The obtained O 18(sucrose/ Vienna Standard Mean Ocean Water (VSMOW)) values are more positive and vary in a wider range (32.1 40.1 parts per thousand) than the O 18(bulk/ VSMOW) values (24.6 27.2 parts per thousand). Furthermore they are shown to depend on the climate parameters maximum day temperature relative air humidity and cloud cover. These findings suggest that O 18(sucrose) of the investigated needles very sensitively reflects the climatically controlled evaporative O 18 enrichment of leaf water and thus highlights the great potential of GC Py IRMS O 18(sucrose) analysis for plant physiology and paleoclimate studies.

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.

Considering the combined effects of CO2 fertilization and climate change drivers on plant physiology leads to a modest increase in simulated European forest transpiration in spite of the effects of CO2-induced stomatal closure. The Earth’s carbon and hydrologic cycles are intimately coupled by gas exchange through plant stomata1,2,3. However, uncertainties in the magnitude4,5,6 and consequences7,8 of the physiological responses9,10 of plants to elevated CO2 in natural environments hinders modelling of terrestrial water cycling and carbon storage11. Here we use annually resolved long-term δ13C tree-ring measurements across a European forest network to reconstruct the physiologically driven response of intercellular CO2 (Ci) caused by atmospheric CO2 (Ca) trends. When removing meteorological signals from the δ13C measurements, we find that trees across Europe regulated gas exchange so that for one ppmv atmospheric CO2 increase, Ci increased by ∼0.76 ppmv, most consistent with moderate control towards a constant Ci/Ca ratio. This response corresponds to twentieth-century intrinsic water-use efficiency (iWUE) increases of 14 ± 10 and 22 ± 6% at broadleaf and coniferous sites, respectively. An ensemble of process-based global vegetation models shows similar CO2 effects on iWUE trends. Yet, when operating these models with climate drivers reintroduced, despite decreased stomatal opening, 5% increases in European forest transpiration are calculated over the twentieth century. This counterintuitive result arises from lengthened growing seasons, enhanced evaporative demand in a warming climate, and increased leaf area, which together oppose effects of CO2-induced stomatal closure. Our study questions changes to the hydrological cycle, such as reductions in transpiration and air humidity, hypothesized to result from plant responses to anthropogenic emissions.

Significant gaps still exist in our knowledge about post-photosynthetic leaf level and downstream metabolic processes and isotopic fractionations. This includes their impact on the isotopic climate signal stored in the carbon isotope composition (δ(13)C) of leaf assimilates and tree rings. For the first time, we compared the seasonal δ(13)C variability of leaf sucrose with intra-annual, high-resolution δ(13)C signature of tree rings from larch (Larix gmelinii Rupr.). The trees were growing at two sites in the continuous permafrost zone of Siberia with different growth conditions. Our results indicate very similar low-frequency intra-seasonal trends of the sucrose and tree ring δ(13)C records with little or no indication for the use of 'old' photosynthates formed during the previous year(s). The comparison of leaf sucrose δ(13)C values with that in other leaf sugars and in tree rings elucidates the cause for the reported (13)C-enrichment of sink organs compared with leaves. We observed that while the average δ(13)C of all needle sugars was 1.2‰ more negative than δ(13)C value of wood, the δ(13)C value of the transport sugar sucrose was on an average 1.0‰ more positive than that of wood. Our study shows a high potential of the combined use of compound-specific isotope analysis of sugars (leaf and phloem) with intra-annual tree ring δ(13)C measurements for deepening our understanding about the mechanisms controlling the isotope variability in tree rings under different environmental conditions.

Journal of Experimental Botany, 66 (19), ISSN:1460-2431, ISSN:0022-0957

How will carbon source-sink relations of 35-yr-old larch trees (Larix decidua) at the alpine treeline respond to changes in atmospheric CO(2) and climate? We evaluated the effects of previously elevated CO(2) concentrations (9 yr, 580 ppm, ended the previous season) and ongoing soil warming (4 yr, + 4°C). Larch branches were pulse labeled (50 at% (13)CO(2)) 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 CO(2) 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 (13)C 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.

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 δ(13)C 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.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.Good peak separation and highly linear and reproducible concentration and δ(13)C 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.The good level of peak separation obtained in this study is reflected in the good precision and linearity of concentration and δ(13)C 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 δ(13)C 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.

[1] The oxygen isotope ratio of precipitation and tree rings is a complex function of climate variables and atmospheric dynamics, which often makes the interpretation of δ18O for palaeoclimate research challenging. Here we analyzed monthly precipitation δ18O series for 1973–2004 and annually resolved tree ring δ18O chronologies for 1945–2004 for three sites in Switzerland: one north of the Alps, one at high-elevation within the Alps, and one south of the Alps. The goal of the study was to improve the understanding of the tree ring archive by a systematic analysis of nonlocal parameters related to atmospheric circulation, in particular, geopotential height field anomalies and the frequency of synoptic weather situations, in addition to the usual local climate parameters like temperature, sunshine duration, and relative humidity. We observed that on average high-pressure situations during summer were associated with relatively high δ18O and low-pressure situations were associated with relatively low δ18O, for both the isotope ratio in precipitation and tree rings. However, correlations to the frequency of weather types were not higher than simple correlations to local temperature. Accordingly, we constructed a combined index from temperature and air pressure that proved to be a good predictor of δ18O in precipitation and used this as the source water term in a tree ring isotope fractionation model. This enabled us to use the model beyond the period where isotope values for precipitation are available, opening new perspectives in the interpretation of long tree ring δ18O chronologies.

In warming climates, soil water content (SWC) may act as an important factor in determining belowground carbon dynamics in boreal forests. Here, we estimated the respiration and nonstructural carbohydrate (NSC) concentrations of tree roots in a mature Scots pine (Pinus sylvestris L.) stand in southern Finland during two growing seasons with contrasting weather conditions. Root respiration was estimated with four different methods: 1) incubating excised roots, 2) partitioning forest floor respirations with root exclusion, or 3) based on temperature response functions and 4) modelling with the whole-tree carbon model 'CASSIA'. In addition, we conducted a drought experiment in a greenhouse to determine the effect of reduced soil-water availability on respiration by incubating soil and roots of Scots pine saplings. We observed that the respiration of incubated roots of Scots pine saplings and soil decreased with drying after excluding the effect of temperature on respiration (RRES), soil being more sensitive to drought than roots. Similarly, RRES of incubated roots in the field was significantly decreased by lowered SWC, whereas respiration of the entire root system estimated with other methods was clearly higher in dryer and warmer than moister and cooler year. Nevertheless, incubated roots excavated from the topsoil are most affected by drying soil, which might not reflect the response of the entire root system. RRES of incubated roots was negatively associated with root fructose and glucose concentrations. At the same time, root fructose, glucose and sucrose concentrations were negatively associated with SWC due to their role in osmoregulation. Thereby it seems that RRES does not directly follow the changes in NSCs despite the apparent correlation. Our study highlights the responsive nature of root carbon dynamics in varying weather events that should be taken into account in estimating and modelling the impacts of warming climate.