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.

The oxygen isotope composition (δ18O) of precipitation is strongly linked to climate and becomes integrated into tree-ring archives. However, this climatic information is only partly preserved in tree rings, as it is modified by hydrological processes prior to root water uptake and by physiological processes before cellulose synthesis. This complicates tree-ring isotope-based climate reconstructions. Nevertheless, direct links between δ18O in precipitation (δ18OP) and tree-ring cellulose (δ18OC) have been rarely tested, largely due to the lack of long-term precipitation δ18O records. Over the past two to three decades, numerous δ18OC chronologies have been established, and they can now be combined with δ18OP data from AI-supported models with high spatiotemporal resolution. This provides a unique opportunity to systematically evaluate the linkage between δ18OP and δ18OC.In this study, we used a network of 45 annually resolved δ18OC chronologies across Europe starting in 1950 and compared them with monthly time series of Piso.AI modelled δ18OP for the corresponding locations. Our main research questions were: (1) which seasonal δ18OP signals are recorded in δ18OC? (2) Is the relationship between δ18OP and δ18OC stable over recent decades? (3) Which factors (species, geography, climate) control the strength of this relationship across the network?We found that correlations between δ18OC and monthly δ18OP were strongest for June, July and August of the current year at most sites. Significant correlations were also observed for other months, including months from the previous year in some cases, but without a consistent pattern across the network. To further examine these relationships, we calculated seasonal and annual mean δ18OP values. Compared with unweighted δ18OP mean values, precipitation-amount weighting reduced correlations with spring, early summer and annual means, thereby narrowing the dominant signal window to the summer period (May–August mean δ18OP). We found that the δ18OP–δ18OC relationship was stable across sites over recent decades, with no systematic change in correlation strength over time. Ongoing analyses use (1) the correlation coefficients (r values) between δ18OC and δ18OP and (2) the δ18O offset between cellulose and precipitation, both considering annual and June-July-August δ18OP values. These metrics are used to investigate the role of species, geography, climate in controlling the observed δ18OP–δ18OC linkage. Our findings improve the understanding of site- and species-specific isotope signal transfer from water sources to tree rings and help identify spatial and temporal climate signals reflected in tree-ring δ18O.

Abstract The position, strength, and variability of precipitation in the South Pacific Convergence Zone (SPCZ) remain challenging for coupled ocean‐atmosphere climate models to represent, leaving future rainfall projections in this dynamically important region uncertain. Here, we present quantitative records of mean annual precipitation (MAP) spanning the past 1,000 years using sediment cores from five freshwater lakes on different islands in the western portion of the SPCZ. Precipitation reconstructions are based on the hydrogen isotope composition of the dinoflagellate biomarker dinosterol, which is inversely correlated with MAP. We show that MAP in the western tropical Pacific increased over the past ∼300 years, with a significant increase beginning in 1,820 ± 75 C.E., coincident with a warming trend in the Western Pacific Warm Pool. These results contrast with the expected drying associated with reduced Pacific Walker Circulation as global temperatures increase in many climate models. Future warming may promote more intense SPCZ precipitation.

The Beaufort Sea region in the Canadian Arctic has undergone substantial sea ice loss in recent decades, primarily driven by anthropogenic climate warming. To place these changes within the context of natural climate variability, Holocene sea ice evolution and environmental conditions (sea surface temperature, salinity, terrestrial input) were reconstructed using lipid biomarkers (HBIs including IP25, OH-GDGT, brGDGT, C16:0 fatty acid, phytosterols) from two marine sediment cores collected from the Beaufort Shelf and slope, spanning the past 9.1 ka and 13.3 cal. kyr BP, respectively. The Early Holocene (11.7-8.2 ka) is characterized by relatively higher sea surface temperature, lower salinity and no spring/summer sea ice until 8.5 ka on the Beaufort Sea slope. Around 8.5 ka, a peak in organic matter content is linked to both increased terrestrial input and primary production and may indicate increased riverine input from the Mackenzie River and terrestrial matter input from coastal erosion. Following this period, terrestrial inputs decreased throughout the Mid-Holocene in both cores. A gradual increase in IP25 and HBI-II concentrations aligns with relatively higher salinity, lower sea surface temperature and rising sea levels, and indicate the establishment of seasonal (spring) sea ice on the outer shelf around 7 ka and on the shelf around 5 ka. These patterns suggest an expansion of the sea ice cover beginning in the Mid-Holocene, influenced by decreasing summer insolation. During the Late Holocene (4.2-1 ka), permanent sea ice conditions are inferred on the slope with a peak during the Little Ice Age. After 1 ka, seasonal sea ice conditions on the slope are observed again, alongside an increase in salinity and terrestrial input, and variable primary productivity. Similar patterns of Holocene sea ice variability have been observed across other Arctic marginal seas, highlighting a consistent response to external climate forcing. Continued warming may drive the Beaufort Sea toward predominantly ice-free conditions, resembling those inferred for the Early Holocene.

Understanding the oxygen and hydrogen isotopic composition (delta 18O and delta 2H values, respectively) of plant source water is fundamental for tracing water uptake in ecohydrological studies. However, discrepancies between cryogenic vacuum distillation (CVD)-extracted soil and xylem water and known plant source water delta 18O and delta 2H values continue to raise concerns about the accuracy of this approach, especially for delta 2H. We conducted two irrigation experiments on juvenile temperate trees to evaluate the accuracy by which CVD soil and xylem water characterize the delta 18O and delta 2H values of plant source water. To do this, we compared the delta 18O and delta 2H of irrigation and mildly vacuum-extracted (MVE) sap water, which has previously been shown to characterize plant source water delta 18O and delta 2H values with high accuracy, with those of CVD-extracted water from soil, xylem and phloem delta 18O and delta 2H values. In both experiments, irrigation water and MVE sap delta 18O and delta 2H values were similar to each other. We yet observed a small and consistent offset between delta 18O and delta 2H values of irrigation water and CVD-extracted soil water delta 18O and delta 2H values. These offsets indicate that CVD soil water captures a mixture of two isotopically distinct soil water pools: a mobile, plant-available pool and an immobile, plant-unavailable pool. Primarily delta 2H values of CVD-extracted xylem water differed from both CVD-extracted water from MVE sap and phloem, while delta 18O differences were minimal. This suggests that artefacts associated with CVD, rather than stem evaporation or phloem-xylem exchange, were the primary source of the observed delta 2H offsets. In summary, our findings demonstrate that CVD is a valid extraction method for accurate plant source water delta 18O values while delta 2H values are associated with artefacts that require correction.

Interpreting tree-ring oxygen isotope composition (δ18Oring) is complicated by the combined influences of source water (δ18Osw) and relative humidity (RH). This study investigates intra- and interannual δ18Oring signals in Scots pine stands in southern and northern Finland over a 10-year period (2010-19). We applied correlation analysis and process-based intra-annual δ18O and δ13C modeling to disentangle RH and δ18Osw signals in δ18Oring. Growth models for xylogenesis were used to date the analyzed tree-ring subsections. Dual isotope modeling provided additional constraints to evaluate the uncertainties caused by xylogenesis. Our results show that δ18Oring signals were dominated by RH, due to its much higher relative variability compared with that of δ18Osw. Generally, correlations were stronger at interannual than intra-annual resolution. Modeling indicated that additional factors complicate the interpretation of intra-annual δ18Oring signals beyond the combined effects of RH and δ18Osw. We show that seasonal variations in the proportion of oxygen exchange with source water during the pathway to tree-ring cellulose may explain the lower RH signal at intra-annual resolution. Incorporating variable oxygen exchange improved model performance and aligned modeled δ18Oring more closely with observations. Despite the encouraging modeling results using growth models for dating tree-ring subsections, we recognize that time integration and alignment will continue to challenge the interpretation of intra-annual isotope signals. Our study demonstrates that combining empirical data analysis with mechanistic modeling is essential for resolving the environmental drivers of δ18Oring and for extending interpretations beyond site-specific conditions. Our findings are particularly relevant as intra-annual δ18O analysis becomes more common, underscoring the importance of time integration and dating tree-ring subsections, highlighting future research needs (e.g., varying oxygen exchange) and advancing their use for climate reconstruction.

Understanding how past climate variability and human activity shaped northern European landscapes is essential for interpreting present-day environmental change. However, high-resolution Holocene records that capture both climatic and anthropogenic signals in small lake catchments remain relatively scarce in the eastern Baltic region. This study addresses that gap by reconstructing Holocene climate and environmental dynamics from the sedimentary material of Lake Pangodi, a semi-closed basin in southern Estonia. We present a multi-proxy reconstruction of Holocene climate and environmental change based on stable isotope geochemistry, sedimentology, pollen, and chironomid records. Stable isotope data document abrupt environmental changes that coincide with regionally recognized early-middle and middle-late Holocene transitions in northern Europe. The early Holocene (similar to 11,700-8200 cal a BP) was characterized by relatively wet conditions and elevated terrigenous sediment influx. This was followed by a drier and more stable middle Holocene (8200-4200 cal a BP) and a more variable late Holocene (4200 cal a BP-present). Chironomid-inferred temperatures indicate warming during the early Holocene and relatively stable warm season temperatures thereafter. Pollen data reveal transitions from early boreal dominance to temperate forest expansion beginning around 8800 cal a BP, followed by a return to more boreal-dominated assemblages after similar to 4800 cal a BP. Increased crop pollen after similar to 850 cal a BP indicates growing human influence on the landscape. These shifts align with regional vegetation chronozones and reflect both climatic and anthropogenic drivers. An abrupt shift to the highest sediment flux at similar to 4200 cal a BP coincides with the transition to the late Holocene, suggesting a regional environmental reorganization that was likely linked to both climatic and landscape changes, including increased hydrological variability and land surface instability.

Microbes are responsible for the cycling of carbon (C) in soils, and predicted changes in soil C stocks under climate change are highly sensitive to shifts in the mechanisms thought to control microbial physiological response to warming. Two mechanisms have been proposed to explain the long-term effects of warming on microbial physiology: microbial thermal acclimation and changes in the quantity and quality of substrates available for microbial metabolism. However, studies disentangling these two mechanisms and assessing how land use affects them are lacking. To disentangle the drivers of changes in microbial physiology in response to long-term climate change, we sampled soils from a 10-year old global change and land use intensity experiment at the Pre-Alpine Terrestrial Environmental Observatories (TERENO project). The global change treatment includes a warming of 2oC and a reduction in precipitation of about 450 mm. We took soil samples at different time-points during the spring season and depths. We performed short-term laboratory incubations over a range of temperatures to measure the relationships between temperature sensitivity of physiology (growth, respiration, carbon use efficiency with the 18O-H2O method) and we characterized the quantity and quality of soil organic matter with the ramped thermal rock-eval pyrolysis at different depths. In this ongoing project, we did not observe thermal acclimation of microbial respiration, growth or CUE to climate change. However, fertilization had the strongest effect on the temperature sensitivity of microbial respiration. In the next steps of this project, we will determine whether climate change and/or land use intensity has an effect on soil organic carbon fractions with different residence times. Our preliminary results show that land use intensity has an overriding effect on the temperature sensitivity of microbial processes compared to long-term climate change.

Climate change, including a reduction in precipitation, increased atmospheric moisture demand, and drying soils, threaten the life-supporting function in trees. In response, trees can exhibit different below-ground drought acclimation strategies, including increased root-water uptake depth and root growth to increase water supply. We initiated a long-term monitoring experiment at the Swiss Canopy Crane II (SCCII) site in Switzerland in 2018, including a rainfall exclusion of 50% during the vegetation period (April-October) since 2023. The SCCII site provides growing conditions representative of a central European mid-mountain range forest and hosts 10 co-occurring European temperate tree species. For six years, we measured the δ 2 H and δ 18 O values of samples collected from tree xylem, soil water in different depths, and precipitation, as well as a multitude of ecophysiological measurements within a great range of environmental conditions (wet and dry), including the exceptionally dry summer in 2023. The extreme conditions in 2023 caused canopy dieback and mortality in individuals of Fagus sylvatica , Picea abies , and Abies alba in the drought treatment indicating critically low soil water supply. We utilized the data to parameterize the hydrological model LWFBrook90.jl with the goal of simulating soil moisture, soil water potential and soil water isotope transport, as well as root water uptake depth under different environmental conditions. By quantifying the temporal origins of root water uptake and running scenarios of further increased drought conditions, we will quantify the access of different tree species to soil water from various depths and the soil water residence time. Moreover, we will quantify how soil water residence time and, thus, the supply of water to tree species at different soil depths varies under different climate change scenarios. First results show that depending on the severity of drought and tree water consumption, the soil water is used up almost entirely within one growing season indicating the vital role of summer precipitation and winter-time refilling. We expect the final results of this study to provide us with valuable insights on soil water retention time and the temporal dynamics of root water uptake under various drought conditions. These findings will increase our understanding critical below-ground drought effects and acclimation of temperate trees.

The stable isotopic composition of plant waxes (δ13C and δ2H values) in lake sediments is a powerful tool for reconstructing past climates and environments, offering critical insights into hydroclimate variability and vegetation dynamics. Different plant types, such as grasses and woody vegetation, can exhibit distinct water sources and isotopic fractionations, directly influencing plant-wax isotopic signatures in sedimentary records. This influence is particularly pronounced in small basins, where local vegetation sources can dominate over regional signals, complicating the interpretation of δ2H records. Disentangling the effects of vegetation changes from hydroclimate variations in lipid biomarkers is paramount for robust interpretations yet remains quite challenging. Here, we aim to quantify the effects of climate, vegetation, and human activities on sedimentary records, improving our understanding of environmental changes in central Switzerland. We utilized δ13C and δ2H records of plant waxes (n-alkanes and fatty acids) from Rotsee, a small lake near Luzern. Our Bayesian-based 14C age model establishes a temporal framework for interpreting changes in vegetation and hydroclimate at Rotsee since the Younger Dryas period (12.9–11.7 ka). The extensive paleoclimate and archaeological research in the region offers a complementary and broader context for our findings. During the Younger Dryas, leaf wax δ13C and δ2H values were very low — consistent with cold and dry conditions. δ2H values of long-chain fatty acids increase rapidly during the transition to the Early Holocene, in what appears to be a double-step change: an initial ~20 ‰ increase driven by hydroclimate changes (δ13C remained stable) followed by ~30 ‰ driven by changes in regional vegetation (accompanied by a ~10‰ δ13C increase). This overall ~50 ‰ increase in δ2H aligns with a major and well-known regional warming trend.During the Holocene Thermal Maximum, around 9.5 to 6 ka, δ13C values peaked, likely reflecting the dominance of woody plants and increased input of aquatic plants, while δ²H values remained stable. However, after 6 ka, there were significant shifts in δ¹³C values, possibly reflecting local vegetation source changes at Rotsee such as a decrease in aquatic plants. Around 2 ka, during the Roman Period, the plant wax isotopic records at Rotsee show a major shift, with δ2H values declining by ~40‰ and δ13C by ~5‰ in long-chain fatty acids. This substantial change in δ2H values exceeds what would be expected based on plausible hydroclimate variations during this period, suggesting a vegetation source-driven change. The intensification of forest clearance and cereal cultivation, as indicated by pollen records, suggests that human-driven land use changes likely strongly influenced the δ2H signal. After medieval times (ca. 0.5 ka), plant wax δ13C and δ2H values increased significantly, likely due to forest regrowth and Rotsee’s eutrophication.Overall, this study highlights the potential of plant-wax isotopes to reveal complex interactions among climate, vegetation, and human activity. Our findings demonstrate how sedimentary records from small basins similar to Rotsee can provide valuable insights into local and regional environmental dynamics.

Phytoplankton play a key role in biogeochemical cycles, impacting atmospheric and aquatic chemistry, food webs, and water quality. However, it remains challenging to reconstruct changes in algal community composition throughout the geologic past, as existing proxies are suitable only for a subset of taxa and/or influenced by degradation. Here, we investigate if compound-specific hydrogen isotope ratios (S2H values) of common algal lipids can serve as (paleo)ecologicalindicators. First, we grew 20 species of algae - representing cyanobacteria, diatoms, dinoflagellates, green algae, and cryptomonads - in batch cultures under identical conditions and measured S2H values of their lipids. Despite identical source water S2H values, lipid S2H values ranged from-455 %o to-52 %o, incorporating variability associated with chemical compound classes and taxonomic groups. In particular, green algae synthesized fatty acids with higher S2H values than other taxa, cyanobacteria synthesized phytol with relatively low S2H values, and diatoms synthesized sterols with higher S2H values than other eukaryotes. Second, we assessed how changes in algal community composition can affect net S2H values of common algal lipids in 20 experimental outdoor ponds, which were manipulated via nutrient loading, and the addition of macrophytes and mussels. High algal biomass in the ponds, which was mainly caused by cyanobacterial and green algal blooms, was associated with higher S2H values for generic fatty acids, relatively stable S2H values for phytol and the dinoflagellate biomarker dinostanol, and lower S2H values for the more cosmopolitan sterol stigmasterol. These results are consistent with expectations from our culture-based analyses, with both datasets indicating large taxon-specific changes that are unlikely to be driven by bacterial heterotrophy. This suggests that measuring S2H values of multiple lipids from sediment and calculating 2 H-offsets between them can resolve changes in algal community composition from changes in source water isotopes. With an appropriate availability of sedimentary lipids, this approach could permit the reconstruction of both taxonomic variability and hydroclimate from diverse sedimentary systems.

Soil water sustains terrestrial life, yet its fate is uncertain under a changing climate. We conducted a deuterium labeling experiment to determine whether elevated atmospheric carbon dioxide (CO 2 ), warming, and drought impact soil water storage and transport in a temperate grassland. Elevated CO 2 created a wetter rootzone compared with ambient conditions, whereas warming decreased soil moisture. Soil water remained well mixed in all global change treatments except for summer drought combined with warming and elevated CO 2 . These combined treatments caused the grassland to conserve water and restricted soil water flow to large, rapidly draining pores without mixing with small, slowly draining pores. Our results suggest that drought in a warmer, more CO 2 -rich climate can severely alter grassland ecohydrology by constraining postdrought soil water flow and grassland water use.

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.

Submerged macrophytes incorporate lake water directly during lipid synthesis, making the δ2H values of their lipids a crucial tool for reconstructing past hydrology and lake states. Despite this significance, the lipid biomarkers of aquatic plants have received less attention compared to terrestrial plants. In particular, in the context of organic geochemical applications, submerged aquatic plants are typically considered as a single group, ignoring potential differences in lipid distributions and hydrogen isotope composition between vascular macrophytes and macroalgae such as charophytes. This gap limits the use of lipid biomarkers in lake sediments to understand past lake water isotopes and vegetation dynamics. In this study, we analyzed the lipid contents and δ2H values of fatty acids, n-alkanes, and the chlorophyll side-chain phytol from paired vascular macrophytes and charophytes collected from 12 oligo-mesotrophic hardwater lakes in northeast Germany. We aim to assess differences between macrophyte groups and their relation to environmental factors and lake water properties, such as lake water δ2H values and pH levels. Our preliminary results reveal a notable predominance of fatty acids over n-alkanes in both macrophyte groups. Vascular macrophytes tended to exhibit a higher, albeit variable, abundance of n-alkanes, fatty acids, and phytol concentrations compared to charophytes. The n-alkanes profiles were mainly comprised of mid to long-chain hydrocarbons (n-C23 to n-C27) and exhibited striking variability among macrophytes. Nonetheless, charophytes were notably characterized by a prominent dominance of n-C27. While the C16:0 fatty acid was the most abundant hydrocarbon in both macrophyte types, vascular macrophytes exhibit a greater abundance of long-chain (C24 to C30) fatty acids. However, our data revealed marked differences in the relative abundance of these long-chained compounds. The overall disparities in the lipid profiles point to distinct lipid biosynthesis pathways or environmental adaptations among the studied aquatic plants. Despite the differences in lipid distributions, no systematic differences were observed in the δ2H values for any studied compound class between the two macrophyte groups, Our results suggest that reconstructions of lake water isotopes based on δ2H values of aquatic plant lipids are unlikely to be influenced by changes in the relative contributions from vascular macrophytes and charophytes.

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.

Variations of oxygen isotopes δ18O in tree rings provide critical insights into past climate and tree physiological processes, yet the mechanisms shaping the intra-annual δ18O signals remain incompletely understood. To address this gap, we investigated how seasonal changes in source water, leaf water, and sugars influence δ18O recorded along the tree rings of Pinus sylvestris in Finland. We conducted a seasonal analysis measuring δ18O from needle water, source water, and phloem sugars and investigated the fraction of oxygen isotope exchange during wood formation. We found that seasonal δ18O amplitudes are significantly reduced from leaf water to tree rings, driven by opposing seasonal patterns in increasing source water δ18O and decreasing evaporative enrichment as relative humidity increases. Additionally, the isotope exchange between source water and phloem sugars further dampens seasonal δ18O signals in the rings. Our findings show that oxygen isotope exchange is critical in shaping δ18O signals, influencing the role of source water and relative humidity recorded on intra-annual resolution. This refined understanding helps interpret tree physiological responses under changing conditions and improves climate reconstructions based on tree rings using intra-annual resolution.

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.

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.