Dr. Marco Lehmann

• Reliable insights from key δ²H bioindicators, n-alkanes and carbohydrates, are hindered by our limited understanding of isotope fractionation processes related to leaf water and primary assimilates. We addressed this with the first study to investigate time-integrated intra-annual δ²H signals of n-alkanes and carbohydrates in a natural forest.
• We sampled 1-yr-old needles (1N) and current-year needles (0N) from five Scots Pine trees in a coniferous forest in Hyytiälä, Finland, biweekly during 2019. The δ²H of their n-alkanes (δ²H_alkane), water-soluble carbohydrates (δ²H_WSC) and starch (δ²H_starch) were evaluated for time-integrated physiological (gas-exchange) and environmental (hydrological) signals.
Time integration was critical for interpreting δ²H_WSC and δ²H_alkane.
• Time-integrated net assimilation rate (T:A_n), the strongest signal in δ²H_WSC, correlated negatively with 1N δ²H_WSC (early season) and positively with 0N δ²H_WSC (late season). Unexpectedly, δ²H_alkane exhibited stronger environmental signals in 1N than in 0N, with the most pronounced being physiologically mediated hydrological signals.
• T:A_n is a major signal in intra-annual δ²H_WSC in a boreal forest, subject to seasonal interactions with needle age and δ²H_starch. There could be enough de novo n-alkane synthesis in situ to enhance the reliability of δ²H_alkane as a hydrological indicator, bringing promise to interpretations of the n-alkane palaeohydrological record.

Stable oxygen (δ¹⁸O) and hydrogen (δ²H) isotope compositions of tree-ring compounds preserve information about environmental waters; however, our understanding of their isotopic relationships is hampered by the lack of long-term data sets. We investigated correlations using unique 17-year (2006–2022) δ¹⁸O and δ²H time series of bi-weekly measured soil solution, modelled precipitation and xylem water, along with those of tree-ring α-cellulose and lignin methoxy groups from Norway spruce (Picea abies) across three Swiss forest sites. We show that tree-ring cellulose δ¹⁸O preserves water source information more effectively than δ²H, making it better suited for ecohydrological reconstructions. We propose δ²H of tree-ring lignin methoxy groups as an alternative proxy for soil water sources, supported by strong correlations where cellulose failed to track soil water isotopes. Significant linear isotopic relationships within and across sites enable the development of transfer functions that link tree-ring to water sources, particularly precipitation and xylem water. We exemplify how these transfer functions can be used to estimate the seasonal origin of water sourced by trees during the growth period. Our findings enhance the interpretation of environmental water isotope signals in tree rings and promote the use of tree-ring isotope-based tools for retrospective retrieval of forest water dynamics.

Aquaporins have long been known to facilitate water transport across membranes inside leaves. However, their role in regulating the water potential (ψ) difference between the cytosol and cell wall has been questioned, as the ψ of the cytosol and cell wall would be in equilibrium under the assumption of water-vapour-saturated leaf intercellular air spaces. Recent advances suggest that intercellular air spaces are unsaturated at high vapour pressure deficit (VPD) and that aquaporins that up-regulate water transport might simultaneously down-regulate CO₂ transport in a competitive manner. Therefore, the currently assumed mechanisms of CO₂ and water transport in the mesophyll under varying VPD must be re-examined. We incorporated the competitive aquaporin hypothesis into the leaf gas exchange pathways with unsaturated intercellular air spaces. We show that the putative competitive control of CO₂- and water-facilitating aquaporins is fully effective only when there is a large ψ gradient between the cytosol and cell wall at high VPD. In this context, the down-regulation of water-facilitating aquaporins and the up-regulation of CO₂-facilitating aquaporins could protect the cytosol from drying and maintain the CO₂ supply for photosynthesis, respectively. While it remains unclear whether unsaturation drives aquaporin activity or vice versa, we identify research challenges that need to be addressed.

Carbon (C) allocation among different plant tissues is crucial for maintaining C balance in forest ecosystems, especially under changing climate conditions. The partitioning of newly assimilated C among plant tissues, interconnected ramets and soil in forests dominated by giant clonal plants, such as moso bamboo (Phyllostachys edulis), and the influence of drought on this partitioning remain poorly understood.
In August 2019, we performed in situ labelling of the entire crown of R0 (ramets that emerged in 2019) of moso bamboo with ¹³CO₂ in plots subjected to a 5-year drought or left untreated (ambient control) in subtropical China. We then traced the ¹³C signatures in the leaves, twigs and fine roots of R0, R1 (ramets that emerged in 2018 and are connected with R0) and R2 (ramets that emerged in 2017 and are connected with R1), as well as in soil organic C (SOC) and soil respiration over the course of 1-year post-labelling.
Drought reduced leaf ¹³C assimilation and its allocation to sink tissues but did not alter the velocity of C transport from source to sink compared to controls. The peak ¹³C signal was observed on day 15 for SOC and on day 5 for respired CO₂ in both drought and ambient control forests. Labelled ¹³C was detected in R1 ramets on day 3 and in R2 on day 7 post-labelling. This study reveals that new assimilates produced by the ‘younger’ R0 ramets are preferentially retained within their own tissues to meet their own demands rather than being allocated to interconnected neighbouring R1 and R2 ramets.
Synthesis. In forests dominated by large clonal plants, such as giant moso bamboo, drought can alter the allocation of newly assimilated C within the tissues of source ramets but may not affect its allocation among interconnected ramets or within plant–soil systems. Our findings highlight the complexity of newly assimilated C partitioning in these forests and suggest that clonal integration may mitigate drought-induced dieback in older ramets through resource sharing under climate change.

To investigate which physiological predispositions led to the drought-induced vitality decline in beech (Fagus sylvatica L.) following the severe 2018 drought, we studied trees with premature leaf discoloration and shedding (early-browning trees) and trees showing no symptoms (vital trees) in a forest in northern Switzerland. We analyzed annual tree-ring width (TRW) and applied a triple isotope approach (i.e., carbon (δ¹³C), oxygen (δ¹⁸O), and hydrogen (δ²H) isotopes) in tree-ring cellulose for the period 1960–2020. To retrieve tree physiological responses, we normalized the tree-ring δ values to temporal isotopic variations in CO₂ or precipitation (Δ). Δ¹³C and Δ¹⁸O values suggest that the early-browning trees had a more conservative water-use strategy and lower stomatal conductance than the vital trees in the initial decades of the measurement period. However, several decades before the onset of crown dieback in 2018, the early-browning trees showed a decrease in TRW and an increase in Δ²H, suggesting a higher use of carbon reserves for the early-browning trees. These long-term trends may be the first signs of a progressive deterioration of the physiology of the early-browning trees. During and after the 2018 drought, changes in Δ²H suggested high carbon investments into drought damage repair for the early-browning trees. Moreover, a higher TRW and isotope sensitivity to previous year’s summer climate in early-browning than vital trees suggests stronger negative carry-over effects. Our findings highlight that the early-browning trees may have already been weakened before the 2018 drought, eventually pushing them beyond their physiological tipping points and inducing dieback.

Rationale: determining several isotope ratios in one analysis multiplies the information that can be retrieved from a sample in a cost-efficient way. The stable isotope ratios of hydrogen (δ²H), carbon (δ¹³C), and oxygen (δ¹⁸O) in organic compounds are highly relevant due to their complimentary hydroclimatic and physiological signals. Different types of organic material reflect different processes and integration times, like short term in leaf sugars and long term in tree ring cellulose, but currently, no simple method exists for their triple isotope analysis.
Methods: here, we present a method that enables the isotopic analyses of the three elements H, C, and O in one run and is applicable to different types of carbohydrates and bulk organic matter. We discuss all steps required from water vapor equilibration necessary for obtaining reliable δ²H values of carbon-bound H to high-temperature conversion (HTC) of the sample to CO and H₂ and to the mass-spectrometric isotope-ratio analysis.
Results: we show that reliable triple isotope analysis is possible for a large range of samples, although it results in some reduction of precision compared to individual isotope analysis. Important considerations are the equilibration procedure, the type of autosampler, selection of HTC reactor, the influence of nitrogen in the sample, the verification of δ¹³C values obtained by HTC versus combustion, and the selection of reference materials.
Conclusions: by presenting a relatively simple triple-isotope method, we promote the use of multi-isotope studies in environmental sciences, which helps in addressing many important climate and ecological research challenges that we face today.

• The hydrogen isotope composition (δ²H) of cellulose is inherently linked to that of sucrose synthesized in leaves. Daytime sucrose is synthesized from triose phosphates produced by the Calvin-Benson-Bassham cycle, while nighttime sucrose is synthesized from remobilized transitory starch in leaves. Photosynthetic metabolism causes starch to be naturally ²H-depleted relative to triose phosphates. Thus, sucrose δ²H values should vary diurnally. However, this has not been tested.
• We made diel measurements of sucrose and starch δ²H in three species differing in their sucrose/starch dynamics (bean, radish and sunflower) under climate controlled and steady-state isotopic conditions.
• Leaf starch was degraded at night and ²H-depleted by around 90‰ compared with daytime sucrose. However, in all tested species we surprisingly observed no effect on nighttime sucrose δ²H and, instead, only species-specific differences. Consequently, the apparent isotope fractionation associated with sucrose biosynthesis (ε_sucrose) was indistinguishable between day and night and within −140‰ to −180‰ across the three species.
• The lack of day/night variation in ε_sucrose could originate from cytosolic sugar metabolism and ²H-enrichment at H-atom positions counteracting the ²H-depletion in starch. Using a simplified steady-state isotopic model of metabolism, we show that differences in day/night fluxes can reduce the expected differences between day/night ε_sucrose. From a practical perspective, this suggests that: (i) estimating ε_sucrose at a single time point might be sufficient to capture δ²H variation under steady-state conditions; and (ii) to extract more than one metabolically sensitive isotope signals from a given compound, position-specific isotope analysis will be required.

• In most tree species, roots serve as major carbon (C) sinks, where C is depleted first when C assimilation is limited. Recent methodological advancements in sugar infusion allow for a better understanding of physiological processes alleviating root C limitation.
• We conducted a glasshouse experiment with maple (Acer pseudoplatanus L.) and pine (Pinus sylvestris L.) saplings that underwent defoliation followed by either slow, fast, or no ¹³C-labeled glucose infusion. We measured photosynthetic parameters, nonstructural carbohydrate (NSC) concentrations, and δ¹³C in cellulose of leaves, twigs, and fine roots, as well as the isotopic composition of dark-respired CO₂.
• Sugar infusion induced photosynthetic downregulation and leaf senescence in maple but not in pine. Leaf photosynthesis was negatively correlated with leaf NSC concentration in maple. These responses exacerbated root C limitation in maple. Conversely, pine maintained stable photosynthetic rates and needle NSC concentrations across treatments, showing the potential of sugar infusion to mitigate root C limitation.
• Our study suggests that exogenous sugar supply reduces the root C availability when it impairs a plant's photosynthetic performance. Species-specific differences influence infused sugar transport and overall source–sink responses. Alleviating C limitation in roots via exogenous sugar addition is feasible only if photosynthesis is not impeded.

Plants’ non-structural carbohydrates (NSCs) serve as their capital for growth, reproduction, defense, and survival. To increase the NSC availability of carbon-limited trees, a recent study revealed the possibility of adding exogenous soluble sugars to carbon-starved trees. This provides an opportunity to investigate carbon allocation between source and sink, as well as the growth and physiological responses to external sugars. Using this method, we infused ¹³C-labeled glucose solution into the stem xylem of sycamore maple (Acer pseudoplatanus L.) trees (Experiment 1) and immersed branch cuttings of various tree species in a ¹³C-labeled glucose solution (Experiment 2). Our aim was to study whether infused sugars contribute to structural growth and how they influence photosynthesis. Specifically, we focused on whether trees can transport and utilize exogenous sugars for growth, and if sugar addition might trigger negative feedback mechanisms on carbon gain. We then traced the ¹³C label in bulk tissue and cellulose, and measured tissue NSC concentrations and leaf photosynthesis. Glucose addition consistently increased leaf NSC concentrations (Experiments 1 and 2), and exogenous sugars added were transported and incorporated into biomass formation in Experiment 1. However, excessive sugar addition triggered a negative feedback response, leading to leaf senescence (Experiments 1 and 2) and reduced photosynthesis (Experiment 2). Our findings validate the recently developed sugar addition method but emphasize the importance of carefully controlling the amount and rate of sugar addition to avoid negative feedback responses. This study will contribute to carbon physiological research, particularly in understanding carbon balance and source-sink relationships at the whole-plant level.

Recurrent climate-driven disturbances impact on the health of European forests that reacted with increased tree dieback and mortality over the course of the last four decades. There is therefore large interest in predicting and understanding the fate and survival of forests under climate change. Forest conditions are monitored within the pan-European ICP Forests programme (UN-ECE International Co-operative Programme on Assessment and Monitoring of Air Pollution Effects on Forests) since the 1980s, with tree crown defoliation being the most widely used parameter. Defoliation is not a cause-specific indicator of tree health and vitality, and there is a need to connect defoliation levels with the physiological functioning of trees. The physiological responses connected to tree crown defoliation are species-specific and concern, among others, water relations, photosynthesis and carbon metabolism, growth, and mineral nutrients of leaves. The indicators to measure physiological variables in forest monitoring programs must be easy to apply in the field with current state-of-the-art technologies, be replicable, inexpensive, time efficient and regulated by ad hoc protocols. The ultimate purpose is to provide data to feed process-based models to predict mortality and threats in forests due to climate change. This study reviews the problems and perspectives connected to the realization of a systematic assessment of physiological variables and proposes a set of indicators suitable for future application in forest monitoring programs.

The analysis of the stable isotopic composition of hydrogen and oxygen in water samples from soils and plants can help to identify sources of vegetation water uptake. This approach requires that the heterogeneous nature of plant and soil matrices is carefully accounted for during experimental design, sample collection, water extraction and analyses. The comparability and shortcomings of the different methods for extracting water and analyzing isotopic composition have been discussed in specialized literature. Yet, despite insightful comparisons of extraction methods and benchmarking methodologies of laboratories worldwide, the community still lacks a roadmap to guide sample collection, extraction, and isotopic analyses, and many practical issues for potential users remain unresolved: for example, which (soil or plant) water pool(s) does the extracted water represent? These constitute a hurdle for the implementation of the approach by newcomers. Here, we summarize discussions led in the framework of the COST Action WATSON ("WATer isotopeS in the critical zONe: from groundwater recharge to plant transpiration"—CA19120). We provide guidelines for (1) sampling soil and plant material for isotopic analysis, (2) methods for laboratory or in situ water extraction, and (3) measurements of isotopic composition. We highlight the importance of considering the process chain as a whole, from experimental design to isotopic analysis to minimize biased estimates of the relative contribution of different water sources to plant water uptake. We conclude by acknowledging some of the limitations of this methodology and advice on the collection of key environmental parameters prior to sample collection for isotopic analyses.

• 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 (e_i), we inferred e_i from oxygen isotope discrimination of CO₂ and water vapour.
• e_i 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 e_i 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 e_i was considered.
• We suggest that the possibility of unsaturated e_i should not be overlooked in measurements related to leaf gas exchange and in stomatal models, especially at high VPD.

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 H₂O 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 (g_s) and net photosynthesis rate (A_n) decoupled across all tested species, with g_s increasing but A_n 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.

Even though they share many thematical overlaps, plant metabolomics and stable isotope ecology have been rather separate fields mainly due to different mass spectrometry demands. New high-resolution bioanalytical mass spectrometers are now not only offering high-throughput metabolite identification but are also suitable for compound- and intramolecular position-specific isotope analysis in the natural isotope abundance range. In plant metabolomics, label-free metabolic pathway and metabolic flux analysis might become possible when applying this new technology. This is because changes in the commitment of substrates to particular metabolic pathways and the activation or deactivation of others alter enzyme-specific isotope effects. This leads to differences in intramolecular and compound-specific isotope compositions. In plant isotope ecology, position-specific isotope analysis in plant archives informed by metabolic pathway analysis could be used to reconstruct and separate environmental impacts on complex metabolic processes. A technology-driven linkage between the two disciplines could allow to extract information on environment–metabolism interaction from plant archives such as tree rings but also within ecosystems. This would contribute to a holistic understanding of how plants react to environmental drivers, thus also providing helpful information on the trajectories of the vegetation under the conditions to come.

Non-structural carbohydrates (NSCs) are building blocks for biomass and fuel metabolic processes. However, it remains unclear how tropical forests mobilize, export, and transport NSCs to cope with extreme droughts. We combined drought manipulation and ecosystem ¹³CO₂ pulse-labeling in an enclosed rainforest at Biosphere 2, assessed changes in NSCs, and traced newly assimilated carbohydrates in plant species with diverse hydraulic traits and canopy positions. We show that drought caused a depletion of leaf starch reserves and slowed export and transport of newly assimilated carbohydrates below ground. Drought effects were more pronounced in conservative canopy trees with limited supply of new photosynthates and relatively constant water status than in those with continual photosynthetic supply and deteriorated water status. We provide experimental evidence that local utilization, export, and transport of newly assimilated carbon are closely coupled with plant water use in canopy trees. We highlight that these processes are critical for understanding and predicting tree resistance and ecosystem fluxes in tropical forest under drought.

The hydrogen isotopic composition (δ²H) of plant compounds is increasingly used as a hydroclimatic proxy; however, the interpretation of δ²H values is hampered by potential coeffecting biochemical and biophysical processes. Here, we studied δ²H values of water and carbohydrates in leaves and roots, and of leaf n-alkanes, in two distinct tobacco (Nicotiana sylvestris) experiments. Large differences in plant performance and biochemistry resulted from (a) soil fertilization with varying nitrogen (N) species ratios and (b) knockout-induced starch deficiency. We observed a strong ²H-enrichment in sugars and starch with a decreasing performance induced by increasing NO₃⁻/NH₄⁺ ratios and starch deficiency, as well as from leaves to roots. However, δ²H values of cellulose and n-alkanes were less affected. We show that relative concentrations of sugars and starch, interlinked with leaf gas exchange, shape δ²H values of carbohydrates. We thus provide insights into drivers of hydrogen isotopic composition of plant compounds and into the mechanistic modeling of plant cellulose δ²H values.

The oxygen and hydrogen isotopic composition (δ¹⁸O, δ²H) of plant tissues are key tools for the reconstruction of hydrological and plant physiological processes and may therefore be used for disentangling reasons of tree mortality. However, how both elements respond to soil drought conditions before death have rarely been investigated.
To test this, we performed a greenhouse study and determined predisposing fertilization and lethal soil drought effects on δ¹⁸O and δ²H values of organic matter (OM) in leaves and tree rings of living and dead saplings of five European tree species. For mechanistic insights, we additionally measured isotopic (i.e., δ¹⁸O and δ²H values of leaf and twig water), physiological (i.e., leaf water potential and gas-exchange) and metabolic traits (i.e., leaf and stem non-structural carbohydrate concentration, C:N ratios).
Across all species, lethal soil drought generally caused a homogenous ²H-enrichment in leaf and tree-ring OM, but a low and heterogenous δ¹⁸O response in the same tissues. Unlike δ¹⁸O values, δ²H values of tree-ring OM were correlated with those of leaf and twig water and with plant physiological traits across treatments and species. The ²H-enrichment in plant OM also went along with a decrease in stem starch concentrations under soil drought compared to well-watered conditions. In contrast, the predisposing fertilization had generally no significant effect on any tested isotopic, physiological, and metabolic traits.
We propose that the ²H-enrichment in the dead trees is related to (i) the plant water isotopic composition, (ii) metabolic processes shaping leaf non-structural carbohydrates, (iii) the use of carbon reserves for growth, and (iv) species-specific physiological adjustments. The homogenous stress imprint on δ²H but not on δ¹⁸O suggests that the former could be used as a proxy to reconstruct soil droughts and underlying processes of tree mortality.

Micro-computed tomography (µCT) is a non-destructive X-ray imaging method used in plant physiology to visualize in-situ plant tissues that enables assessments of embolized xylem vessels. Whereas evidence for X-ray-induced cellular damage has been reported, the impact on plant physiological processes such as carbon (C) uptake, transport, and use are unknown. Yet, these damages could be particularly relevant for studies that track embolism and C fluxes over time. We examined the physiological consequences of µCT-scanning for xylem embolism over three months by monitoring net photosynthesis (A_net), diameter growth, chlorophyll concentration (Chl), and foliar non-structural carbohydrate (NSC) content in four deciduous tree species: hedge maple (Acer campestre), ash (Fraxinus excelsior), European hornbeam (Carpinus betulus), and sessile oak (Quercus petraea). C transport from the canopy to the roots was also assessed through ¹³C labelling. Our results show that monthly X-ray application did not impact foliar A_net, Chl, NSC content, and C transport. Although X-ray effects did not vary between species, the most pronounced impact was observed in sessile oak, marked by stopped growth and stem deformations around the irradiated area. The absence of adverse impacts on plant physiology for all the tested treatments indicates that laboratory-based µCT systems can be used with different beam energy levels and doses without threatening the integrity of plant physiology within the range of tested parameters. However, the impacts of repetitive µCT on the stem radial growth at the irradiated zone leading to deformations in sessile oak might have lasting implications for studies tracking plant embolism in the longer-term.

The effects of rising atmospheric CO₂ concentrations (C_a) with climate warming on intrinsic water-use efficiency and radial growth in boreal forests are still poorly understood. We measured tree-ring cellulose δ¹³C, δ¹⁸O, and tree-ring width in Larix dahurica (larch) and Betula platyphylla (white birch), and analyzed their relationships with climate variables in a boreal permafrost region of northeast China over past 68 years covering a pre-warming period (1951–1984; base period) and a warm period (1985–2018; warm period). We found that white birch but not larch significantly increased their radial growth over the warm period. The increased intrinsic water-use efficiency in both species was mainly driven by elevated C_a but not climate warming. White birch but not larch showed significantly positive correlations between tree-ring δ¹³C, δ¹⁸O and summer maximum temperature as well as vapor pressure deficit in the warm period, suggesting a strong stomatal response in the broad-leaved birch to temperature changes. The climate warming-induced radial growth enhancement in white birch is primarily associated with a conservative water-use strategy. In contrast, larch exhibits a profligate water-use strategy. It implies an advantage for white birch over larch in the warming permafrost regions.