Dry inside: progressive unsaturation within leaves with increasing vapour pressure deficit affects estimation of key leaf gas exchange parameters.

Climate change not only leads to higher air temperatures but also increases the vapour pressure deficit (VPD) of the air. Understanding the direct effect of VPD on leaf gas exchange is crucial for precise modelling of stomatal functioning. We conducted combined leaf gas exchange and online isotope discrimination measurements on four common European tree species across a VPD range of 0.8-3.6 kPa, while maintaining constant temperatures without soil water limitation. In addition to applying the standard assumption of saturated vapour pressure inside leaves (ei), we inferred ei from oxygen isotope discrimination of CO2 and water vapour. ei desaturated progressively with increasing VPD, consistently across species, resulting in an intercellular relative humidity as low as 0.73 ± 0.11 at the highest tested VPD. Assuming saturation of ei overestimated the extent of reductions in stomatal conductance and CO2 mole fraction inside leaves in response to increasing VPD compared with calculations that accounted for unsaturation. In addition, a significant decrease in mesophyll conductance with increasing VPD only occurred when the unsaturation of ei was considered. We suggest that the possibility of unsaturated ei should not be overlooked in measurements related to leaf gas exchange and in stomatal models, especially at high VPD.

Uncoupling of stomatal conductance and photosynthesis at high temperatures: mechanistic insights from online stable isotope techniques.

The strong covariation of temperature and vapour pressure deficit (VPD) in nature limits our understanding of the direct effects of temperature on leaf gas exchange. Stable isotopes in CO2 and H2 O vapour provide mechanistic insight into physiological and biochemical processes during leaf gas exchange. We conducted combined leaf gas exchange and online isotope discrimination measurements on four common European tree species across a leaf temperature range of 5-40°C, while maintaining a constant leaf-to-air VPD (0.8 kPa) without soil water limitation. Above the optimum temperature for photosynthesis (30°C) under the controlled environmental conditions, stomatal conductance (gs ) and net photosynthesis rate (An ) decoupled across all tested species, with gs increasing but An decreasing. During this decoupling, mesophyll conductance (cell wall, plasma membrane and chloroplast membrane conductance) consistently and significantly decreased among species; however, this reduction did not lead to reductions in CO2 concentration at the chloroplast surface and stroma. We question the conventional understanding that diffusional limitations of CO2 contribute to the reduction in photosynthesis at high temperatures. We suggest that stomata and mesophyll membranes could work strategically to facilitate transpiration cooling and CO2 supply, thus alleviating heat stress on leaf photosynthetic function, albeit at the cost of reduced water-use efficiency.

Updating the dual C and O isotope-Gas-exchange model: A concept to understand plant responses to the environment and its implications for tree rings.

The combined study of carbon (C) and oxygen (O) isotopes in plant organic matter has emerged as a powerful tool for understanding plant functional responses to environmental change. The approach relies on established relationships between leaf gas exchange and isotopic fractionation to derive a series of model scenarios that can be used to infer changes in photosynthetic assimilation and stomatal conductance driven by changes in environmental parameters (CO2 , water availability, air humidity, temperature, nutrients). We review the mechanistic basis for a conceptual model, in light of recently published research, and discuss where isotopic observations do not match our current understanding of plant physiological response to the environment. We demonstrate that (1) the model was applied successfully in many, but not all studies; (2) although originally conceived for leaf isotopes, the model has been applied extensively to tree-ring isotopes in the context of tree physiology and dendrochronology. Where isotopic observations deviate from physiologically plausible conclusions, this mismatch between gas exchange and isotope response provides valuable insights into underlying physiological processes. Overall, we found that isotope responses can be grouped into situations of increasing resource limitation versus higher resource availability. The dual-isotope model helps to interpret plant responses to a multitude of environmental factors.

Plant responses to rising vapor pressure deficit.

Recent decades have been characterized by increasing temperatures worldwide, resulting in an exponential climb in vapor pressure deficit (VPD). VPD has been identified as an increasingly important driver of plant functioning in terrestrial biomes and has been established as a major contributor in recent drought-induced plant mortality independent of other drivers associated with climate change. Despite this, few studies have isolated the physiological response of plant functioning to high VPD, thus limiting our understanding and ability to predict future impacts on terrestrial ecosystems. An abundance of evidence suggests that stomatal conductance declines under high VPD and transpiration increases in most species up until a given VPD threshold, leading to a cascade of subsequent impacts including reduced photosynthesis and growth, and higher risks of carbon starvation and hydraulic failure. Incorporation of photosynthetic and hydraulic traits in 'next-generation' land-surface models has the greatest potential for improved prediction of VPD responses at the plant- and global-scale, and will yield more mechanistic simulations of plant responses to a changing climate. By providing a fully integrated framework and evaluation of the impacts of high VPD on plant function, improvements in forecasting and long-term projections of climate impacts can be made.

Effect of Vapor Pressure Deficit on Gas Exchange in Wild-Type and Abscisic Acid-Insensitive Plants.

Stomata control the gas exchange of terrestrial plant leaves, and are therefore essential to plant growth and survival. We investigated gas exchange responses to vapor pressure deficit (VPD) in two gray poplar (Populus × canescens) lines: wild type and abscisic acid-insensitive (abi1) with functionally impaired stomata. Transpiration rate in abi1 increased linearly with VPD, up to about 2 kPa. Above this, sharply declining transpiration was followed by leaf death. In contrast, wild type showed a steady or slightly declining transpiration rate up to VPD of nearly 7 kPa, and fully recovered photosynthetic function afterward. There were marked differences in discrimination against 13CO2 (Δ13C) and C18OO (Δ18O) between abi1 and wild-type plants. The Δ13C indicated that intercellular CO2 concentrations decreased with VPD in wild-type plants, but not in abi1 plants. The Δ18O reflected progressive stomatal closure in wild type in response to increasing VPD; however, in abi1, stomata remained open and oxygen atoms of CO2 continued to exchange with 18O enriched leaf water. Coupled measurements of Δ18O and gas exchange were used to estimate intercellular vapor pressure, e i In wild-type leaves, there was no evidence of unsaturation of e i, even at VPD above 6 kPa. In abi1 leaves, e i approached 0.6 times saturation vapor pressure before the precipitous decline in transpiration rate. For wild type, a sensitive stomatal response to increasing VPD was pivotal in preventing unsaturation of e i In abi1, after taking unsaturation into account, stomatal conductance increased with increasing VPD, consistent with a disabled active response of guard cell osmotic pressure.

The 18 O-signal transfer from water vapour to leaf water and assimilates varies among plant species and growth forms.

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.

The effect of 18 O-labelled water vapour on the oxygen isotope ratio of water and assimilates in plants at high humidity.

Our understanding of how temporal variations of atmospheric water vapour and its isotopic composition (δ18 OV ) influence water and assimilates in plants remains limited, restricting our ability to use δ18 O as a tracer of ecophysiological processes. We exposed oak (Quercus robur) saplings under wet and dry soil moisture conditions to 18 O-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 δ18 OV into water and assimilates (e.g. sucrose, hexoses, quercitol and starch) in the leaf lamina, main veins and twigs over 24 h. The immediate δ18 OV effect was highest for δ18 O of leaf lamina water, but 40% lower on δ18 O of main vein water. To a smaller extent, we also observed changes in δ18 O of twig xylem water. Depending on the individual assimilation rate of each plant, the 18 O-label was partitioned among different assimilates, with highest changes in δ18 O of starch/sucrose and lowest in δ18 O of quercitol. Additionally, 18 O-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 δ18 O signal is incorporated into biomarkers.

Oxygen isotopes in tree rings are less sensitive to changes in tree size and relative canopy position than carbon isotopes.

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.

Oxygen isotope fractionations across individual leaf carbohydrates in grass and tree species.

Almost no δ18 O data are available for leaf carbohydrates, leaving a gap in the understanding of the δ18 O relationship between leaf water and cellulose. We measured δ18 O values of bulk leaf water (δ18 OLW ) and individual leaf carbohydrates (e.g. fructose, glucose and sucrose) in grass and tree species and δ18 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 (εbio ) of more than 27‰ relative to δ18 OLW , which might be explained by isotopic leaf water and sucrose synthesis gradients. δ18 OLW and δ18 O values of carbohydrates and cellulose in grasses were strongly related, indicating that the leaf water signal in carbohydrates was transferred to cellulose (εbio  = 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 δ18 OLW but not if modelled directly from δ18 O of individual carbohydrates. We conclude that δ18 OLW is not always a good substitute for δ18 O of synthesis water due to isotopic leaf water gradients. Thus, compound-specific δ18 O analyses of individual carbohydrates are helpful to better constrain (post-)photosynthetic isotope fractionation processes in plants.

Plasticity in gas-exchange physiology of mature Scots pine and European larch drive short- and long-term adjustments to changes in water availability.

Adjustment mechanisms of trees to changes in soil-water availability over long periods are poorly understood, but crucial to improve estimates of forest development in a changing climate. We compared mature trees of Scots pine (Pinus sylvestris) and European larch (Larix decidua) growing along water-permeable channels (irrigated) and under natural conditions (control) at three sites in inner-Alpine dry valleys. At two sites, the irrigation had been stopped in the 1980s. We combined measurements of basal area increment (BAI), tree height and gas-exchange physiology (Δ13 C) for the period 1970-2009. At one site, the Δ13 C of irrigated pine trees was higher than that of the control in all years, while at the other sites, it differed in pine and larch only in years with dry climatic conditions. During the first decade after the sudden change in water availability, the BAI and Δ13 C of originally irrigated pine and larch trees decreased instantly, but subsequently reached higher levels than those of the control by 2009 (15 years afterwards). We found a high plasticity in the gas-exchange physiology of pine and larch and site-specific responses to changes in water availability. Our study highlights the ability of trees to adjust to new conditions, thus showing high resilience.

Warm season precipitation signal in δ2 H values of wood lignin methoxyl groups from high elevation larch trees in Switzerland.

RATIONALE: In this study, we tested stable hydrogen isotope ratios of wood lignin methoxyl groups (δ2 Hmethoxyl values) as a palaeoclimate proxy in dendrochronology. This is a quite new method in the field of dendrochronology and the sample preparation is much simpler than the methods used before to measure δ2 H values from wood. METHODS: We measured δ2 Hmethoxyl values in high elevation larch trees (Larix decidua Mill.) from Simplon Valley (southern Switzerland). Thirty-seven larch trees were sampled and five individuals analysed for their δ2 Hmethoxyl values at annual (1971-2009) and pentadal resolution (1746-2009). The δ2 Hmethoxyl values were measured as CH3 I released upon treatment of the dried wood samples with hydroiodic acid. 10-90 µL from the head-space were injected into the gas chromatography/high-temperature conversion/isotope ratio mass spectrometry (GC/HTC-IRMS) system. RESULTS: Testing the climate response of the δ2 Hmethoxyl values, the annually resolved series show a positive correlation of r = 0.60 with June/July precipitation. The pentadally resolved δ2 Hmethoxyl series do not show any significant correlation to climate parameters. CONCLUSIONS: Increased precipitation during June and July, which are on average warm and relatively dry months, results in higher δ2 H values of the xylem water and, therefore, higher δ2 H values in the lignin methoxyl groups. Therefore, we suggest that δ2 Hmethoxyl values of high elevation larch trees might serve as a summer precipitation proxy.

Oxygen isotope analysis of levoglucosan, a tracer of wood burning, in experimental and ambient aerosol samples.

RATIONALE: Levoglucosan is formed from cellulose during biomass burning. It is therefore often used as a specific tracer to quantify the contribution of wood burning to the aerosol loading. The stable oxygen isotope composition (δ18 O value) of biomass is determined by the water cycle and varies regionally, and hence the δ18 O value of levoglucosan could help to identify source regions of organic aerosols. METHODS: After solvent extraction of the organic fraction and concentration steps, a recently developed methylation derivatisation technique was applied on experimental (i.e. controlled wood-burning experiments) and on ambient aerosol samples from Switzerland and Lithuania. The method achieves sufficient compound separation for isotope analysis in atmospheric particulate matter, enabling δ18 O analysis of levoglucosan by gas chromatography/pyrolysis-isotope ratio mass spectrometry (GC/Pyr-IRMS), with a precision better than 1.0 ‰ and an accuracy of 0.3 ‰. RESULTS: The δ18 O value of the levoglucosan released during controlled wood-burning experiments was not significantly different from the cellulose δ18 O values, which implies very little or no isotope fractionation during wood burning under the given conditions. While the δ18 O values of levoglucosan in Swiss samples were as expected for the source region, those in Lithuania were 1-4 ‰ lower than expected. This may be due to differences in vegetation (grass vs wood) or burning conditions (high vs low temperatures). CONCLUSIONS: Low oxygen isotope fractionation between cellulose and levoglucosan and clear differences in levoglucosan δ18 O values between the Swiss and Lithuanian ambient samples demonstrate that our new method is useful for source appointment studies on wood-burning-derived aerosols.

Inferring foliar water uptake using stable isotopes of water.

A growing number of studies have described the direct absorption of water into leaves, a phenomenon known as foliar water uptake. The resultant increase in the amount of water in the leaf can be important for plant function. Exposing leaves to isotopically enriched or depleted water sources has become a common method for establishing whether or not a plant is capable of carrying out foliar water uptake. However, a careful inspection of our understanding of the fluxes of water isotopes between leaves and the atmosphere under high humidity conditions shows that there can clearly be isotopic exchange between the two pools even in the absence of a change in the mass of water in the leaf. We provide experimental evidence that while leaf water isotope ratios may change following exposure to a fog event using water with a depleted oxygen isotope ratio, leaf mass only changes when leaves are experiencing a water deficit that creates a driving gradient for the uptake of water by the leaf. Studies that rely on stable isotopes of water as a means of studying plant water use, particularly with respect to foliar water uptake, must consider the effects of these isotopic exchange processes.

The impact of an inverse climate-isotope relationship in soil water on the oxygen-isotope composition of Larix gmelinii in Siberia.

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.

A dynamic leaf gas-exchange strategy is conserved in woody plants under changing ambient CO2 : evidence from carbon isotope discrimination in paleo and CO2 enrichment studies.

Rising atmospheric [CO2 ], ca , is expected to affect stomatal regulation of leaf gas-exchange of woody plants, thus influencing energy fluxes as well as carbon (C), water, and nutrient cycling of forests. Researchers have proposed various strategies for stomatal regulation of leaf gas-exchange that include maintaining a constant leaf internal [CO2 ], ci , a constant drawdown in CO2 (ca  - ci ), and a constant ci /ca . These strategies can result in drastically different consequences for leaf gas-exchange. The accuracy of Earth systems models depends in part on assumptions about generalizable patterns in leaf gas-exchange responses to varying ca . The concept of optimal stomatal behavior, exemplified by woody plants shifting along a continuum of these strategies, provides a unifying framework for understanding leaf gas-exchange responses to ca . To assess leaf gas-exchange regulation strategies, we analyzed patterns in ci inferred from studies reporting C stable isotope ratios (δ(13) C) or photosynthetic discrimination (∆) in woody angiosperms and gymnosperms that grew across a range of ca spanning at least 100 ppm. Our results suggest that much of the ca -induced changes in ci /ca occurred across ca spanning 200 to 400 ppm. These patterns imply that ca  - ci will eventually approach a constant level at high ca because assimilation rates will reach a maximum and stomatal conductance of each species should be constrained to some minimum level. These analyses are not consistent with canalization toward any single strategy, particularly maintaining a constant ci . Rather, the results are consistent with the existence of a broadly conserved pattern of stomatal optimization in woody angiosperms and gymnosperms. This results in trees being profligate water users at low ca , when additional water loss is small for each unit of C gain, and increasingly water-conservative at high ca , when photosystems are saturated and water loss is large for each unit C gain.

A novel methylation derivatization method for δ(18)O analysis of individual carbohydrates by gas chromatography/pyrolysis-isotope ratio mass spectrometry.

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.

Malate as a key carbon source of leaf dark-respired CO2 across different environmental conditions in potato plants.

Dissimilation of carbon sources during plant respiration in support of metabolic processes results in the continuous release of CO2. The carbon isotopic composition of leaf dark-respired CO2 (i.e. δ (13) C R ) shows daily enrichments up to 14.8‰ under different environmental conditions. However, the reasons for this (13)C enrichment in leaf dark-respired CO2 are not fully understood, since daily changes in δ(13)C of putative leaf respiratory carbon sources (δ (13) C RS ) are not yet clear. Thus, we exposed potato plants (Solanum tuberosum) to different temperature and soil moisture treatments. We determined δ (13) C R with an in-tube incubation technique and δ (13) C RS with compound-specific isotope analysis during a daily cycle. The highest δ (13) C RS values were found in the organic acid malate under different environmental conditions, showing less negative values compared to δ (13) C R (up to 5.2‰) and compared to δ (13) C RS of soluble carbohydrates, citrate and starch (up to 8.8‰). Moreover, linear relationships between δ (13) C R and δ (13) C RS among different putative carbon sources were strongest for malate during daytime (r(2)=0.69, P≤0.001) and nighttime (r(2)=0.36, P≤0.001) under all environmental conditions. A multiple linear regression analysis revealed δ (13) C RS of malate as the most important carbon source influencing δ (13) C R . Thus, our results strongly indicate malate as a key carbon source of (13)C enriched dark-respired CO2 in potato plants, probably driven by an anapleurotic flux replenishing intermediates of the Krebs cycle.

Comparison of δ(18)O and δ(13)C values between tree-ring whole wood and cellulose in five species growing under two different site conditions.

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.

Lack of photosynthetic or stomatal regulation after 9 years of elevated [CO2] and 4 years of soil warming in two conifer species at the alpine treeline.

Alpine treelines are temperature-limited vegetation boundaries. Understanding the effects of elevated [CO2 ] and warming on CO2 and H2 O gas exchange may help predict responses of treelines to global change. We measured needle gas exchange of Larix decidua Mill. and Pinus mugo ssp. uncinata DC trees after 9 years of free air CO2 enrichment (575 µmol mol(-1) ) and 4 years of soil warming (+4 °C) and analysed δ(13) C and δ(18) O values of needles and tree rings. Tree needles under elevated [CO2 ] showed neither nitrogen limitation nor end-product inhibition, and no down-regulation of maximal photosynthetic rate (Amax ) was found. Both tree species showed increased net photosynthetic rates (An ) under elevated [CO2 ] (L. decidua: +39%; P. mugo: +35%). Stomatal conductance (gH2O ) was insensitive to changes in [CO2 ], thus transpiration rates remained unchanged and intrinsic water-use efficiency (iWUE) increased due to higher An . Soil warming affected neither An nor gH2O . Unresponsiveness of gH2O to [CO2 ] and warming was confirmed by δ(18) O needle and tree ring values. Consequently, under sufficient water supply, elevated [CO2 ] induced sustained enhancement in An and lead to increased C inputs into this ecosystem, while soil warming hardly affected gas exchange of L. decidua and P. mugo at the alpine treeline.

Soil warming alters microbial substrate use in alpine soils.

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.