The oxygen (δ18O) and hydrogen (δ2H) isotope compositions of leaf and xylem water shape tree-ring isotope baselines, while the fraction of sugars undergoing isotopic modification downstream of leaves (fO, fH) determines the dominant hydrologic signal. However, limited information on the seasonal dynamics of these isotope sources and on the drivers of f variation constrains tree-ring isotope interpretation. We measured intra-annual δ18O and δ2H in stem water, sugar, starch, and tree-ring α-cellulose of beech and spruce over two growing seasons. Using modelled leaf water δ18O and δ2H, we estimated seasonal f values and examined their relationships with nonstructural carbohydrate concentrations and climate. Tree rings primarily recorded δ18O and δ2H signatures of leaf water, despite seasonal changes in fO and fH. We found no clear transfer of intra-annual xylem water isotopic signals into sugars, starch, or cellulose. Seasonal fO and fH can be negatively correlated. Both were related to climate variables, but only fO was correlated with nonstructural carbohydrate concentrations. Thus, isotopic fractionation downstream of leaves does not always override the seasonal imprint of leaf water in tree rings. These findings provide insight into the controls on the fO-fH covariation, supporting more robust interpretations of climate variability from tree-ring isotope records.

RATIONALE:The stable hydrogen isotope composition (δ2H) of plant compounds can serve as environmental or metabolic proxies, but interpretations are hindered by insufficient mechanistic understanding. This can be improved by analyzing the δ2H values of metabolic intermediates, such as sucrose, which is the direct substrate of cellulose and a main transport sugar. However, current preparation methods for carbohydrates in general and sucrose in particular are not time-efficient. METHODS:We evaluated methods that use acetylation of soluble carbohydrate plant extracts to aid in purification as well as isotopic analysis of plant sugars such as sucrose. Extracts were obtained using either hot water or hot 80% ethanol. Acetylated extracts were then purified using an established liquid-liquid separation (LL) method or a new solid phase extraction (SPE) method that we developed. We also evaluated glucose produced from starch after enzymatic digestion. Method performance was evaluated based on quantified yields and the impact on measured δ2H values. RESULTS:Acetylated sucrose and starch-derived glucose were sufficiently resolved for gas chromatography in all cases. No isotopic biasing was detected for any method. Acetylated sucrose yields differed among methods, with 80% ethanol resulting in approximately threefold higher extraction yield compared to water, and SPE giving smaller but still sufficient yields compared to LL. Sample throughput was doubled with the SPE method compared to the LL method, which allows for larger batch sizes compared to LL. CONCLUSIONS:We developed an efficient method to analyze compound-specific plant carbohydrate δ2H values using gas chromatography-isotope ratio mass spectrometry (GC-IRMS). This can be applied in experiments aimed at investigating the processes that shape cellulose δ2H values, including deconvoluting the metabolic and hydro-climatic sources of isotopic variation.

Although the hydrogen (δ2H) and oxygen (δ18O) isotopic signature of tree rings is dependent on the environmental water, such as precipitation and soil water that trees have taken up (i.e. “source water”), estimating the spatio-temporal origin of water sources through analysis of water stable isotopes in tree rings is not a straightforward approach. This is because 1) our knowledge on the contribution and the variability of individual isotopic fractionation steps between source water and tree rings is limited, and 2) in situ measurements that consider the seasonality of the isotopic composition of source water and cellulose synthesis are rare. Within the framework of the EU Cost Action WATSON (#CA19120 - WATer isotopeS in the critical zONe), we analyzed (1) δ2H and δ18O in tree-ring cellulose and stem sugar, (2) δ2H and δ18O in soil water at shallower (15 cm) and deeper (80 cm) depths in up to bi-weekly resolution and (3) modelled isotopic variations in precipitation, soil water, stem xylem water, and leaf water using mechanistic and process-based models for three long-term forest monitoring sites in Switzerland over 20 years. We used this data to explain intra-annual (2021-2022) and inter-annual (2003-2022) δ2H and δ18O variations in tree-ring cellulose of beech (Fagus sylvatica) and spruce (Picea abies). At the intra-annual scale, preliminary findings indicate a pronounced isotopic enrichment in the second half of the growing season and marked seasonal variations in the isotopic composition of soil water at shallower depths compared to deeper layers. However, such fluctuations were strongly dampenend in the intra-annual δ2H and δ18O variations observed in the tree-ring cellulose and stem sugars of both tree species, which may indicate the use of deeper soil water sources or scrambling of the source water isotope signal because of isotope fractionation before cellulose synthesis. In further analyses at the inter-annual scale, we will investigate how well δ2H and δ18O in tree rings can function as indicators of source water through time-window correlation analysis between water and tree-ring stable isotopes and comparisons between measured and modelled data. Our study aims to enhance models of hydrogen and oxygen isotope fractionation. This will improve the use of both elements in tree rings as innovative ecohydrological proxies for retrospectively reconstructing environmental water sources.

Forests experience complex light environments, but the detailed roles of photosynthetic, stomatal, mesophyll, and biochemical responses to dynamic blue light remain unclear in tree species. We measured the blue-light responses of leaf gas exchange, online isotope discrimination, photorespiration, and chlorophyll fluorescence in grey alder (Alnus incana) and holm oak (Quercus ilex), and investigated the underlying biochemical and physiological mechanisms. With increasing blue light, differing photosynthetic and stomatal responses consistently led to a decrease in intrinsic water-use efficiency (iWUE) in the two species. For alder, the decline in iWUE was primarily due to a reduced photosynthesis rate (An); for oak, although An also decreased, blue-light-stimulated stomatal opening played a major role. Although the reduction in An was linked to blue-light-induced photoprotective processes in alder, it was coordinated with mesophyll conductance (gm) in both species. The maximum carboxylation rate of Rubisco and gm imposed considerable photosynthetic limitations, especially at high blue-light levels. However, the component of gm that responded to blue light and coordinated with An was the chloroplast membrane in alder whereas it was the cell wall and plasma membrane in oak. Our findings highlight species-specific physiological strategies in the response to blue light and underscore the importance of considering spectral composition when assessing carbon-water trade-offs in forest trees.

The intricate architecture of plant metabolic networks and the dynamic fluxes of elements through these networks are fundamental determinants of how carbon (C) is partitioned among growth, reproduction, storage, respiration and the synthesis of secondary metabolites. While these C fluxes are critical to cellular function and plant life, their routine measurement remains a significant challenge. This review aimed to highlight the substantial potential of hydrogen (H) isotopes of plant carbohydrates to bridge this methodological gap by serving as a flux‐based proxy for primary C metabolism. This potential is demonstrated from both a theoretical perspective and by summarising available evidence at the whole‐molecule and position‐specific levels. The utility of this proxy is significant for understanding species' metabolic plasticity, assessing plant responses to environmental change and selecting superior metabolic phenotypes in agriculture and forestry. However, for this proxy to be fully realised, several fundamental questions remain. This includes the identification of specific metabolic reactions associated with isotopic variation and their relationship to plant performance. We outline several approaches to advance the development of an H‐isotope based plant metabolic proxy for plant performance.

The oxygen isotope composition of cellulose (δ18O values) has been suggested to contain information on stomatal conductance (gs) responses to rising pCO2. The extent by which pCO2 affects leaf water and cellulose δ18O values (δ18OLW and δ18OC) and the isotope processes that determine pCO2 responses of gs in δ18OLW and δ18OC are, however, unknown. We tested the effects of pCO2 on gs, δ18OLW and δ18OCin a greenhouse experiment, where six herbaceous plant species were grown under pCO2 levels ranging from 200 to 500 ppm. An increase in pCO2 caused a decline in gs. The effects of gs on δ18OLW were caused by direct and indirect mechanisms but were generally small. The model parameter effective path length (Lm) was unaffected by changes in pCO2. pCO2 effects on δ18OLW were not directly transferred to plant δ18OC but were attenuated in grasses and amplified in dicotyledonous herbs and legumes. This is likely because of functional group specific pCO2 effects on the model parameter pxpex. Our study removes critical uncertainties for using δ18OC as a proxy for gs. At the same time, our study shows that gs effects on δ18OLW and δ18OC are rather small, possibly too small to be detected in natural settings.

The hydrogen isotopic composition of organic compounds carries information relating to the isotopic composition of biosynthetic source water, as well as source-organism biochemistry. This has led to diverse applications in areas such as paleoclimatology, ecology, and criminal forensics. Yet, measurement poses a unique isotopic challenge because hydrogen bound to oxygen or nitrogen can exchange with ambient water or vapor, unlike the hydrogen that is bound to carbon. This creates a need to account for this so-called exchangeable hydrogen. In some cases, this can be done by permanent replacement via chemical derivatization, but this is often not convenient or even possible. This has led to the development of dual water equilibration methods in which the exchangeable hydrogen in a sample is equilibrated with water with a known isotopic composition in a controlled manner as the last step in sample preparation prior to measurement. Dual water equilibration methods have facilitated applications in a range of subdisciplines, especially for applications focused on plant carbohydrate-rich materials such as cellulose and bulk wood, and on keratin in animal migration and ecology. The term “exchangeable hydrogen” has generally been used inconsistently in environmental applications. In some cases, the term is used to describe only the hydrogen that can freely exchange with ambient vapor at room temperature conditions, while in other cases the term directly refers to all hydrogen that is not covalently bound to carbon and can therefore theoretically undergo isotopic exchange. These two definitions are inconsistent with one another because in many biomolecules, such as cellulose and keratin, a large portion of the hydrogen that is not carbon-bound is engaged in hydrogen bonding and is important for the macromolecular structure of the material. This bridging hydrogen, although not carbon-bound, is more difficult to isotopically exchange, and has the potential to be excluded by some types of dual water equilibration approaches. As a consequence, the fraction of hydrogen that is measured as exchangeable varies between sample types and methodologies, resulting in different hydrogen isotope values. In this study we compared hydrogen isotope values after dual water equilibrations on plant carbohydrates and animal keratins using two different analytical approaches, one of which targeted only the freely exchangeable hydrogen pool, and the other of which targeted the theoretically exchangeable hydrogen pool. For all sample types, we observed large differences in the calculated fraction of exchangeable hydrogen, with the freely exchangeable approach yielding exchange rates 10-15 % smaller than those from the theoretically exchangeable approach. The data also showed a greater range of hydrogen isotope values for the approach that achieved higher degrees of hydrogen exchange, suggesting that the range in bridging hydrogen isotope values among samples was lower than that of carbon-bound hydrogen. We suggest modification of the term “exchangeable” in dual water equilibration studies to indicate whether the freely or the potentially exchangeable hydrogen is being targeted, and therefore the extent to which the bridging hydrogen has been isotopically exchanged.

The intricate architecture of plant metabolic networks and the dynamic fluxes of elements through these networks are fundamental determinants of how carbon (C) is partitioned among growth, reproduction, storage, respiration and the synthesis of secondary metabolites. While these C fluxes are critical to cellular function and plant life, their routine measurement remains a significant challenge. This review aimed to highlight the substantial potential of hydrogen (H) isotopes of plant carbohydrates to bridge this methodological gap by serving as a flux-based proxy for primary C metabolism. This potential is demonstrated from both a theoretical perspective and by summarising available evidence at the whole-molecule and position-specific levels. The utility of this proxy is significant for understanding species' metabolic plasticity, assessing plant responses to environmental change and selecting superior metabolic phenotypes in agriculture and forestry. However, for this proxy to be fully realised, several fundamental questions remain. This includes the identification of specific metabolic reactions associated with isotopic variation and their relationship to plant performance. We outline several approaches to advance the development of an H-isotope based plant metabolic proxy for plant performance.

Understanding changes in the physiological responses of trees to disturbances, and establishing proxies to reconstruct past events, is of high importance in a changing world. Recent studies have demonstrated the potential of δ2H in tree-ring cellulose as a proxy for physiological changes in carbon utilization, reflecting shifts between the use of current assimilates and stored C sources. These findings might explain the considerable annual variations in the strength of the δ18O and δ2H (O-H) relationship despite the shared hydrological pathway, underlining the complex interaction of hydrological and physiological processes. One of the situations where there is a clear disruption of carbon assimilation and tree functioning is defoliation events. Thus, tree-ring isotopes can be utilized to test the physiological signal recorded in tree rings by quantifying changes in δ13C, δ2H and δ18O values, and the decoupling of the O-H relationship. Here, we investigated the isotopic fingerprint of abiotic and biotic defoliation events in tree-ring cellulose, including (i) late-spring frost on European beech near its upper elevational limit in the Swiss Jura(ii) pine processionary moth outbreaks in northern Italy, and (iii) cockchafer moth outbreaks on archaeological oak material from Central European lowlands. Across all defoliation types, a common fingerprint was identified with significantly enriched δ2H, depleted δ18O, resulting in the decoupled (negative) O-H relationship, and non-affected δ13C values. As defoliation causes reduced fresh carbon assimilation, the remobilization of stored non-structural carbohydrates (NSC) is likely the fundamental process for plant growth, metabolism, and canopy re-flushing. NSC differ in their isotopic ratio compared to fresh photosynthates, by exhibiting 2H-enrichment and 18O-depletion, explaining the negative O-H relationship in tree-ring cellulose. Since defoliation has been shown to induce allocation shifts by prioritizing NSC storage over radial growth, foliage loss also leads to substantial secondary growth reductions which was observed across all defoliation types. The generally non-significant changes in δ13C between outbreak and non-outbreak years indicate minor impacts on leaf stomatal conductance. In conclusion, this common isotopic fingerprint provides valuable insight into past defoliation events and their reconstruction, which is particularly relevant in the context of rapid environmental change.

When you grow plants in the light, the hydrogen isotopic composition (δ2H) of plant compounds such as cellulose show lower δ2H values (are 2H-depleted) relative to plants grown heterotrophically in the dark. Therefore, it is logical to assume that photosynthetic reactions introduce 2H-depleted hydrogen atoms into carbohydrates. But where in the C reductive pathway (Calvin-Benson-Bassham cycle, CBB) does this occur? Or more interestingly, can we interpret the degree of 2H-depletion of plant compounds with respect to this key reaction(s)? With the recent resurgence of studies offering hydrogen isotopes as a new proxy for plant central carbon and energy metabolism, such a fundamental question seems pertinent to answer. We 1) examine the stereospecific mechanism of hydride transfer via NADP(H) catalyzed by oxidoreductases (ferredoxin-NADP+ reductase, glyceraldehyde 3-phosphate dehydrogenase) as a key reason why photoproduced NADPH is not directly the source of 2H-depletion of autotrophically produced carbohydrates, 2) reconcile the site-specific deuterium abundance pattern differences between C3 and C4 (NADP-ME) species of hydrogen bound to position C-4 in glucose, and 3) urge greater investment in position-specific and complimentary metabolomic analyses to progress the development of hydrogen isotopes as a metabolic proxy.

High-resolution carbon isotope ratio (δ13C) measurements of tree rings have the potential to provide seasonal environmental information. However, due to the complexity of the wood formation processes, the reliability of this method for intra-seasonal reconstruction of growing conditions remains unclear. We therefore investigated the intra-annual variation of δ13C in tree rings of three conifer species (Pinus sylvestris L., Picea abies (L.) H. Karst., Abies alba Mill.) across sites from the Swiss Alps to assess their response to seasonal variation of soil water potential (SWP) and vapour pressure deficit (VPD). Intra-annual δ13C values at a resolution of 10 points per year were assessed using laser-ablation isotope-ratio mass spectrometry. Seasonal δ13C patterns were analysed for synchronicity across trees and species, and their correlation with on-site environmental variables was used to determine the driving factors of δ13C, to reconstruct growing-season dynamics, and to estimate the timings of the growth dynamics and the allocation of carbon to xylem formation. The δ13C patterns showed high synchronicity between species, with characteristic maxima in wet and dry years occurring in the middle of the ring and at the end of the ring, respectively. Seasonal δ13C variations reliably reflected atmospheric dryness. Higher than normal soil dryness hindered the integration of further fresh assimilates into the xylem, thus allowing the identification of species- and site-specific threshold conditions that disrupt wood formation. The δ13C of Scots pine shows the strongest correlations with VPD and SWP, making it an excellent indicator of environmental variability. Silver fir appeared to integrate carbon into xylem structural material over a longer season than the other conifers, whilst Norway spruce shows more plastic, site-specific responses to environmental conditions. In conclusion, we identify how atmospheric and soil drought jointly impact tree growth and intra-annual δ13C patterns across conifer species, offering valuable insights for climate reconstructions and wider applications in forest dynamics.

RATIONALE:The hydrogen stable isotope composition (δ 2H) of organic samples carries information about processes including hydrology, climate, ecology, and plant metabolism. Numerous methods exist to control for hydrogen in organic samples that can isotopically exchange with ambient water or vapor. METHODS:We compared two established dual water steam equilibration approaches to control for exchangeable hydrogen in isotopic analysis: hot vacuum equilibration using the Uniprep autosampler and hot atmospheric pressure equilibration under varying equilibration temperatures and times. We evaluated how the extent of hydrogen isotope exchange and calculated exchange-corrected hydrogen stable isotope values varied among methods and sample types. RESULTS:More exchange could be achieved with atmospheric pressure methods compared to the Uniprep, and we confirmed that this was not a drying artifact. When direct comparisons were possible, atmospheric pressure methods yielded δ 2H values that agreed better with values from chemical derivatization, while Uniprep values agreed better with values provided for purchased reference materials based on room temperature equilibrations. Best long-term precision was achieved with fitted rather than prescribed isotope fractionation factors for equilibration. CONCLUSIONS:Different equilibration methods can give reproducible yet distinct results. This is due to varying degrees to which different methods access non-carbon-bound hydrogen that is difficult to liberate from interior hydrogen bonds in complex biological structures such as cellulose or keratins. The optimal method depends on the application.

Summary Significant variation in plant organic compound hydrogen stable isotope (δ2H) values among species from a single location suggests species biochemistry diversity as a key driver. However, the biochemical mechanisms and the biological relevance behind this species‐specific δ2H variation remain unclear. We analyzed δ2H values of cellulose and n‐alkanes across 179 eudicot species in a botanical garden sampled in 2019, and cellulose, n‐alkanes, fatty acids and phytol δ2H values from 56 eudicot species sampled in 2020. We utilized the observed species variation in δ2H values to determine phylogenetic structure and mechanistic constraints for biochemical 2H‐fractionation. A strong phylogenetic signal in lipid compound δ2H values implies that the drivers of species variation in lipid δ2H values are evolutionarily conserved. By contrast, species variation in cellulose δ2H values was not strongly linked to phylogeny. Generally low‐explanatory power of relationships between δ2H values of different compounds (R2 < 0.26) implies nonubiquitous drivers of species variation in plant organic compound δ2H values. Historically, variable biochemical 2H‐fractionation was often attributed to δ2H values of H incorporated from NADPH. Instead, the results from this study suggest that species variation in biochemical 2H‐fractionation largely occurs independently within biosynthetic pathways. For lipids, these mechanisms appear strongly linked to evolutionary history.

Plant cellulose hydrogen (H) stable isotope compositions (δ2H) integrate hydrological and biochemical information, and therefore measurements from archives such as tree rings can be valuable for understanding past climate and plant metabolic responses to environmental change. Although the hydrological component that is integrated into cellulose δ2H values is relatively well understood, the biochemical reactions that can alter δ2H values of metabolites used for cellulose biosynthesis remain cryptic. Attempts at establishing models to simplify the interpretation of cellulose δ2H values have been made, like the widely used cellulose δ2H model by Roden et al. (2000) using the terms quantified by Yakir & DeNiro (1990). However, independent quantification of the parameters in this model, and assessment of their variability with respect to plant C metabolism, has been limited. The cellulose δ2H model uses the δ2H compositions of leaf water and source water, autotrophic and heterotrophic 2H-fractionation (εA and εH, respectively), and the proportion of carbon (C) bound H that exchanges with xylem water during cellulose biosynthesis (ƒ) to explain variation in cellulose δ2H values. By growing plants along a gradient of source water δ2H values under autotrophic and heterotrophic conditions, the original, εA, εH, and ƒ were determined for the aquatic plant Lemna gibba L.. One drawback of this approach is that it assumes these terms are the same when plants are grown in the light vs the dark. We recently reassessed the model for terrestrial plants by measuring δ2H values of leaf sucrose and found species variation in εA (Holloway-Phillips et al., 2022), but were unable to resolve variation associated with ƒ and εH. In the present experiment we assessed a new experimental approach to quantify all model parameters for autotrophically grown plants using regression analysis. This required growing plants with variation in the isotopic offset between xylem water and leaf water (∆LW) and measuring sucrose and cellulose δ2H values from leaves and roots. In a previous study we determined that mutation-induced inhibition of starch synthesis in leaves resulted in higher cellulose δ2H values compared with the wildtype, which was hypothesized to occur preceding sucrose synthesis in the leaves (Baan et al., 2023). Using this new approach, we tested whether this effect was indeed mostly established in source cells during de novo sucrose synthesis (εA), or was a result of 2H-fractionating processes in sink cells prior to cellulose synthesis (εH and ƒ). Preliminary analyses show an increase in leaf sucrose δ2H values in the mutant relative to the wild type, implying that εA is also dependent on plant C metabolism within a given species.

Summary statement We provide evidence that photosynthetically produced NADPH is not the major source of 2 H‐depletion in carbohydrates.

In this opinion paper, we provide an overview of the difference between evaporative and carboxylation sites in leaves, and explain consequences in particular for water isotopes at natural abundance.

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.

The hydrogen (δ²H) and oxygen (δ¹⁸O) isotopic signatures of tree rings depend on that of the environmental water sources, such as precipitation and soil water, taken up by trees (i.e., "source water"). Analyzing δ²H and δ¹⁸O of tree rings is thus a promising approach for reconstructing the spatio-temporal origins of tree water sources. However, such reconstructions remain rare, likely due to methodological challenges, including the analysis of hydrogen isotopes in tree rings and the availability of historical source water isotope data.In this study, we present a first attempt to reconstruct the temporal origins of water used by trees during the 20th century (1901–1995) with annually resolved tree-ring δ¹⁸O time series. The reconstruction is based on a δ¹⁸O chronology of whole wood, sampled from the latewood of spruce (Picea abies) at Bettlachstock, Switzerland. Our choice of site and species reflects a conservative approach, as a transfer function linking δ¹⁸O of tree-ring cellulose to the δ¹⁸O of source waters (e.g., stem xylem water and soil solutions) was recently established over a 17-year period (2006–2022) at the same site. After accounting for the isotopic offset between whole wood and cellulose, we estimated δ¹⁸O values of soil solution (80 cm depth) and stem xylem water during the growing season (May–September) using a linear transfer function. Further, using modeled precipitation δ¹⁸O data and the estimated δ¹⁸O of soil solution and xylem water, we deduced interannual variations in the seasonal origin index (SOI) of soil solution and xylem water during the 20th century. Our results show that the reconstructed δ¹⁸O values and SOI of xylem water were higher than those of soil solutions, suggesting a greater contribution of summer water to xylem water than to soil solutions. Interestingly, while conditions from 1900 to 1970 remained relatively stable, we observed abrupt increases in SOI for both soil solutions and stem xylem water between 1970 and 1995. These recent changes were not due to an increase in summer precipitation amount but may be linked to shifts in seasonal precipitation patterns, causing a relative increase in the contribution of summer precipitation in tree water sources. Despite these findings, uncertainties in precipitation isotope data and transfer functions need further investigation to draw more definitive conclusions. We hope this study will stimulate discussion on the advances and limitations of using tree-ring isotopes to reconstruct historical water sources.

Stable carbon isotopes are a powerful tool to study photosynthesis. Initial applications consisted of determining isotope ratios of plant biomass using mass spectrometry. Subsequently, theoretical models relating C isotope values to gas exchange characteristics were introduced and tested against instantaneous online measurements of 13C photosynthetic discrimination. Beginning in the twenty-first century, laser absorption spectroscopes with sufficient precision for determining isotope mixing ratios became commercially available. This has allowed collection of large data sets at lower cost and with unprecedented temporal resolution. More data and accompanying knowledge have permitted refinement of 13C discrimination model equations, but often at the expense of increased model complexity and difficult parametrization. This chapter describes instantaneous online measurements of 13C photosynthetic discrimination, provides recommendations for experimental setup, and presents a thorough compilation of equations available to researchers. We update our previous 2018 version of this chapter by including recently improved descriptions of (photo)respiratory processes and associated fractionations. We discuss the capabilities and limitations of the diverse 13C discrimination model equations and provide guidance for selecting the model complexity needed for different applications.

Scots pine (Pinus sylvestris L.) is a common European tree species, and understanding its acclimation to the rapidly changing climate through physiological, biochemical or structural adjustments is vital for predicting future growth. We investigated a long-term irrigation experiment at a naturally dry forest in Switzerland, comparing Scots pine trees that have been continuously irrigated for 17 years (irrigated) with those for which irrigation was interrupted after 10 years (stop) and non-irrigated trees (control), using tree growth, xylogenesis, wood anatomy, and carbon, oxygen and hydrogen stable isotope measurements in the water, sugars and cellulose of plant tissues. The dendrochronological analyses highlighted three distinct acclimation phases to the treatments: irrigated trees experienced (i) a significant growth increase in the first 4 years of treatment, (ii) high growth rates but with a declining trend in the following 8 years and finally (iii) a regression to pre-irrigation growth rates, suggesting the development of a new growth limitation (i.e. acclimation). The introduction of the stop treatment resulted in further growth reductions to below-control levels during the third phase. Irrigated trees showed longer growth periods and lower tree-ring δ13 C values, reflecting lower stomatal restrictions than control trees. Their strong tree-ring δ18 O and δ2 H (O-H) relationship reflected the hydrological signature similarly to the control. On the contrary, the stop trees had lower growth rates, conservative wood anatomical traits, and a weak O-H relationship, indicating a physiological imbalance. Tree vitality (identified by crown transparency) significantly modulated growth, wood anatomical traits and tree-ring δ13 C, with low-vitality trees of all treatments performing similarly regardless of water availability. We thus provide quantitative indicators for assessing physiological imbalance and tree acclimation after environmental stresses. We also show that tree vitality is crucial in shaping such responses. These findings are fundamental for the early assessment of ecosystem imbalances and decline under climate change.