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Recent methodological advancements in determining the nonexchangeable hydrogen isotopic composition (δ2Hne) of plant carbohydrates make it possible to disentangle the drivers of hydrogen isotope (2H) fractionation processes in plants. Here, we investigated the influence of phylogeny on the δ2Hne of twig xylem cellulose and xylem water, as well as leaf sugars and leaf water, across 73 Northern Hemisphere tree and shrub species growing in a common garden. 2H fractionation in plant carbohydrates followed distinct phylogenetic patterns, with phylogeny reflected more in the δ2Hne of leaf sugars than in that of twig xylem cellulose. Phylogeny had no detectable influence on the δ2Hne of twig or leaf water, showing that biochemistry, not isotopic differences in plant water, caused the observed phylogenetic pattern in carbohydrates. Angiosperms were more 2H-enriched than gymnosperms, but substantial δ2Hne variations also occurred at the order, family, and species levels within both clades. Differences in the strength of the phylogenetic signals in δ2Hne of leaf sugars and twig xylem cellulose suggest that the original phylogenetic signal of autotrophic processes was altered by subsequent species-specific metabolism. Our results will help improve 2H fractionation models for plant carbohydrates and have important consequences for dendrochronological and ecophysiological studies., New Phytologist, 239 (2), ISSN:0028-646X, ISSN:1469-8137

Recent methodological achievements in determining the non-exchangeable hydrogen isotopic composition (δ2Hne) of non-structural carbohydrates such as sugars allow to disentangle of so far hidden hydrogen isotope (2H) fractionation processes influencing δ2Hne of plant carbohydrates. We conducted two climate chamber experiments to have a closer look at the basic biochemical drivers of the photosynthetic 2H fractionation between water and sugar and the post-photosynthetic 2H fractionation between sugars and cellulose in leaves: First, we studied the impact of the different biochemical reactions in 10 species with C3, 7 species with C4, and 8 species with CAM carbon fixation pathways, and their response to changes in temperature and vapor pressure deficit (VPD). Second, we investigated the impact of a temperature increase from 10 to 40°C in 5°C steps under a constant VPD on leaf level photosynthesis and metabolic functioning of 7 plant species. The first experiment revealed distinct differences in the photosynthetic 2H fractionation between C3, C4, and CAM plants. In addition, the observed intensity and direction of the shifts in δ2Hne in response to changes in temperature and VPD in C3 plants was species specific, absent in C4 plants, and again species-specific in CAM plants. However, post-photosynthetic 2H fractionation was very similar among the three types of carbon fixation. We demonstrate that, in contrary to widespread believes, the 2H enrichment during post-photosynthetic 2H fractionation is driven by the carbohydrate metabolism, and not by an isotopic exchange with surrounding water. The results of the second experiment identified a plants’ metabolic activity, and its response to changes in temperature, as a major driver of the post-photosynthetic 2H fractionation of leaf sugars in C3 species. Our results clearly demonstrate that δ2Hne of plant carbohydrates are driven by plants metabolism and its response to the environment, which are species-specific. This will help to improve our current ability to interpret δ2Hne chronologies in tree rings and other plant archives, and to use 2H fractionation in carbohydrates as a novel proxy to study a plants’ metabolic properties.

The analysis of a Europe-wide network of tree-ring stable isotopes has shown that the climatic signal of δ2H in tree-ring cellulose (C6H10O5), is far weaker compared to those recorded in carbon (δ13C) and oxygen (δ18O)isotopes. Furthermore, the δ2H and δ18O relationships were shown to be site dependent and significantly deviated from the Global Meteoric Water Line. These results suggest that non-climatic effects are modifying the hydrological signature of δ2H. Recent experiments have underlined the potential of δ2H in tree-ring cellulose as a physiological indicator of shifts in autotrophic versus heterotrophic processes. However, the impact of these processes has not yet been quantified under natural conditions.Defoliating insect outbreaks can disrupt photosynthetic production and carbon allocation, stimulating the remobilization of stored carbohydrates. Such disturbance events, therefore, provide unique opportunities to evaluate the impact of changes in the use of fresh versus stored non-structural carbohydrates, i.e., of non-climatic signals stored in δ2H. By exploring a 700-year tree-ring record from Switzerland, we assess the impact of 79 larch budmoth (LBM, Zeiraphera griseana) outbreaks on the growth of its Larix decidua host trees.LBM outbreaks significantly altered the tree-ring isotopic signature, creating a 2H-enrichment and a depletion in 18O 13C. Changes in tree physiology during outbreak years are shown by the decoupling of δ2H and δ18O (O–H relationship), in contrast to the positive correlation in non-outbreak years. The O–H relationship in outbreak years was not significantly affected by temperature, indicating that non-climatic physiological processes dominate over climate in determining δ2H variations. We conclude that the combination of these isotopic parameters may serve as a metric for assessing changes in physiological mechanisms over time and that hydrogen isotopes can be considered as a proxy for non-climatic disturbance signals in dendrochronological research.

Stable isotope compositions of carbon (δ13C) and oxygen (δ18O) in plant carbohydrates such as photosynthetic assimilates or cellulose are widely applied tools to reconstruct climate and plant physiological responses. In contrast, applications of hydrogen isotope composition (δ2H) in plant carbohydrates are limited because of previous methodological constrains and limited knowledge on processes causing hydrogen isotope fractionations. To better understand the individual climatic drivers of isotopic variations in tree rings, particularly for δ2H, we performed a controlled experiment over one growing season in climate chambers with saplings of broadleaf and conifer tree species. The growing conditions resembled conditions that can be typically found in the field: vapor pressure deficit (VPD; 1.0, 1.6, and 2.2 kPa), air temperature (T; 25 and 30 °C), and soil drought (D, well-watered and extreme dry). After 5 months of treatment, δ13C, δ18O, and δ2H of water, sugars and starch in stems and leaves, as well as in cellulose of the recent year tree rings were measured. For δ2H analyses of plant carbohydrates, we applied a newly developed hot water vapour equilibration method (Schuler et al., 2022, doi.org/10.1111/pce.14193). Across all species, first results show that the three elements in tree-ring cellulose respond differently to the climatic drivers: both δ2H and δ13C values increased with increases in D and VPD, but the VPD responses were more pronounced under high than under low T conditions. In contrast, δ18O values were affected by T and VPD, while the VPD response was more pronounced under wet than under dry soil D conditions. Thus, the combination of δ2H and δ13C values could be used to identify D occurrences independent of VPD conditions, while δ18O value is a better indicator for T and VPD responses. In the following steps, the isotopic variations in tree-ring cellulose will be linked to those in water, sugars, and starch and their concentrations in leaf and stem material, as well as to various other structural and functional traits which have been measured throughout the experiment (Schönbeck et al., 2022, doi.org/10.1111/pce.14425). With this unique experimental design, we aim to provide new knowledge facilitating the interpretation of stable isotope patterns in tree rings under field conditions.

Recent studies have challenged the interpretation of plant water isotopes obtained through cryogenic vacuum distillation (CVD) based on observations of a large 2H fractionation. These studies have hypothesized the existence of an H-atom exchange between water and organic tissue during CVD extraction with the magnitude of H exchange related to relative water content of the sample; however, clear evidence is lacking. Here, we systematically tested the uncertainties in the isotopic composition of CVD-extracted water by conducting a series of incubation and rehydration experiments using isotopically depleted water, water at natural isotope abundance, woody materials with exchangeable H, and organic materials without exchangeable H (cellulose triacetate and caffeine). We show that the offsets between hydrogen and oxygen isotope ratios and expected reference values (Δ2H and Δ18O) have inversely proportional relationships with the absolute amount of water being extracted, i.e. the lower the water amount, the higher the Δ2H and Δ18O. However, neither Δ2H nor Δ18O values, were related to sample relative water content. The Δ2H pattern was more pronounced for materials with exchangeable H atoms than with non-exchangeable H atoms. This is caused by the combined effect of H exchange during the incubation of materials in water and isotopic enrichments during evaporation and sublimation that depend on absolute water amount. The H exchange during CVD extraction itself was negligible. Despite these technical issues, we observed that the water amount-dependent patterns were much less pronounced for samples at natural isotope abundance and particularly low when sufficiently high amounts of water were extracted (>600 µL). Our study provides new insights into the mechanisms causing isotope fractionation during CVD extraction of water. The methodological uncertainties can be controlled if large samples of natural isotope abundance are used in ecohydrological studies.

Temperature (T) and vapour pressure deficit (VPD) are important drivers of plant hydraulic conductivity, growth, mortality, and ecosystem productivity, independently of soil water availability. Our goal was to disentangle the effects of T and VPD on plant hydraulic responses. Young trees of Fagus sylvatica L., Quercus pubescens Willd. and Quercus ilex L. were exposed to a cross-combination of a T and VPD manipulation under unlimited soil water availability. Stem hydraulic conductivity and leaf-level hydraulic traits (e.g., gas exchange and osmotic adjustment) were tracked over a full growing season. Significant loss of xylem conductive area (PLA) was found in F. sylvatica and Q. pubescens due to rising VPD and T, but not in Q. ilex. Increasing T aggravated the effects of high VPD in F. sylvatica only. PLA was driven by maximum hydraulic conductivity and minimum leaf conductance, suggesting that high transpiration and water loss after stomatal closure contributed to plant hydraulic stress. This study shows for the first time that rising VPD and T lead to losses of stem conductivity even when soil water is not limiting, highlighting their rising importance in plant mortality mechanisms in the future., Plant, Cell & Environment, 45 (11), ISSN:0140-7791, ISSN:1365-3040

While carbon (δ13C) and oxygen (δ18O) isotopes in tree-ring cellulose are widely used as climatic and physiological proxy in dendro sciences, the processes that are affecting the fractionation of non-exchangeable hydrogen (δ2H) isotopes and shaping the tree-ring δ2H profile are barely understood and thus not widely applied yet.To establish a first comprehensive comparison of the photosynthetic and post-photosynthetic 2H-fractionation of northern-hemisphere trees, we sampled leaves and twigs of 152 trees representing 73 species, 48 genus, 19 families and 12 orders containing both evergreen and deciduous angio- and gymnosperms in a common garden, as well as diurnal cycles (6 species from 6 families) of leaf sugar. We extracted leaf water and sugar, as well as twig water and the current year twig xylem cellulose for δ2H analysis. Leaf sugar and twig cellulose were measured with a newly established hot water vapour equilibration method.Our findings show a wide variation in 2H-fractionation between species growing at a common site. The measured δ2H values ranged from -63.5 to –33.4‰ for xylem water, between -22.3 and +28.5‰ for leaf water, between -160.9 and +12.6‰ for leaf sugar and between -79.1 and +6.9‰ for twig cellulose. The biological fractionation between leaf water and leaf sugar ranged between -169.6 and +24.2‰ and between leaf sugar and current year twig cellulose from -34.6 to +116.8‰. In general, sugar and cellulose of gymnosperms were significantly more 2H depleted than those of angiosperm species, with no impact of the leaf shedding behaviour to the measured δ2H values. We observed significant differences in the δ2H values between different orders and families, but not between genus and species within a family or genus, respectively. This pattern indicates that the photosynthetic and post-photosynthetic 2H-fractionation are based on conservative metabolic reactions with a generally low mutation rate.Additionally, the results from our diurnal sampling of leaf sugar are showing first evidence for a dynamic and species-specific nature of the photosynthetic 2H-fractionation, which is in contrast to current models, which are assuming the same constant 2H-fractionation processes for all plant species.We conclude that the here presented results will help to improve our understanding of the mechanisms influencing the δ2H values of leaf sugar and tree-ring cellulose and thus enabling the scientific community to use δ2H in tree-ring cellulose as the third isotope-proxy for dendrochronological studies.

Recent studies suggest that isotope ratios of the carbon-bound non-exchangeable hydrogen (δ2H) in plant cellulose and lipids can indicate changes in the primary carbon and energy metabolism; however, systematic investigations are scarce.Here, we studied δ2H patterns in two different tobacco (N. sylvestris) model systems, where severe changes in the plant primary metabolism were known: 1) along a nitrogen (N) supply gradient and 2) in a starch-less knockout mutant (pgm). Specifically, we measured δ2H of water, bulk soluble sugars, transitory starch, and cellulose in leaves and roots, using a novel hot water vapor equilibration method and TC/EA-IRMS. Besides, we measured δ2H values of leaf n-alkanes with GC-IRMS.We observed clear δ2H differences in sugars and starch along the N gradient and a 2H-enrichment of both assimilates in pgm compared to a wild type control. The photosynthetic 2H-fractionation between leaf water and sugars/starch reached a maximum of ca. 100‰ in both model systems and was related to changes in concentrations of primary metabolites (e.g. sugars, starch, organic and amino acids), enzymatic activities, gas-exchange, and growth. The signal of the primary carbon metabolism was also visible in δ2H of leaf and root cellulose in both system, but dampened compared to those of sugars and starch. In contrast, the signal was absent in leaf n-alkanes in both systems.Our results provide the first direct evidence that changes in the primary leaf carbon metabolism are imprinted on δ2H of plant carbohydrates in leaf and roots. The metabolic signal might therefore be reconstructed from plant material of important paleo archives (e.g. tree-ring cellulose, lake sediments) and help to better understand plant-climate interactions. The absence of the signal in δ2H of leaf n-alkanes is surprising and suggests a strong difference in metabolic fluxes between carbohydrates and lipids. Yet, this observation may help to further disentangle the processes shaping hydrogen isotopes in plants.

Recent studies challenge the use of plant water from cryogenic vacuum distillation (CVD) extraction in accurately representing the hydrogen and oxygen isotopic composition (δ2H and δ18O) of plant source water. This is hypothesized to be because the δ2H in extracted water depends on tissue relative water content (RWC), which might be explained by the exchange of H-atoms between water and organic material. Secondary hypotheses focus on extraction artefacts related to evaporation and sublimation, but clear evidence is lacking. Here, we hypothesized that the observed δ2H and δ18O offsets (Δ2H and Δ18O) are influenced by (i) an H-exchange effect, (ii) tissue water amount or RWC and (iii) evaporation and sublimation enrichments.The hypotheses were systematically tested by three corresponding experiments. Firstly, we added a range of strongly depleted reference water (δ2H: ca. -460‰; δ18O: ca. -170‰; 50–1200 μl) to organic materials (with and without exchangeable H) of constant weight (200 mg), followed by a 24 h incubation. In addition, the same range of pure reference water and tap water without any material were used as controls. Secondly, we incubated dry stem segments (Larix decidua) of different sizes in excess of reference water for 24 hours, then they were took out for extracting known water contents from samples with known RWC. Accordingly, fresh twig segments from the same species were prepared for extracting water with natural abundance. Thirdly, a range of different amounts of reference water (50–1200 μl) was added directly into the water collection tubes of the CVD extraction system. In addition, 2 ml glass vials containing the same range of reference water amounts were incubated in a climate chamber at 25 °C and 50 % relative humidity with lids open for 2 hours. All the samples, except the water in the glass vials, were extracted using a standard CVD extraction method for 2 hours. We found that both Δ2H and Δ18O values were not related to changes in RWC. In contrast, we observed an inversely proportional relationships with water amount, i.e., the lower the water amount, the higher the Δ2H and Δ18O. For Δ2H, the pattern was more pronounced for materials with exchangeable H, which reached 150‰ at the lowest water amount and decreased to -20‰ with increasing water amounts when the depleted reference water was used. However, the pattern was much less pronounced for the samples with natural isotopic abundance, indicating that the magnitude of the pattern is probably dependent on isotope ratios of plant water and water vapour in the laboratory. The evaporation and sublimation tests both showed that the pattern was partly caused by an increasing isotopic enrichment with decreasing water amount.In conclusion, we identified a significant artefact of CVD when water is present in small amounts, particularly when δ2H and δ18O of the water was below natural isotope abundances. We therefore recommend extracting > 600 μl of water. Moreover, we provide first evidence of a significant H-exchange effect, suggesting that using hydrogen isotopes for estimating plant source water will remain challenging in future.

The hydrogen isotopic signature (δ2H) of water in trees contains information on plant functional responses to climatic changes and on the origin of the water. This is also true for the non-exchangeable hydrogen isotopic signature (δ2HNE) of plant organic matter, which contains additional physiological and biochemical information that can be dated to specific years if extracted from annual rings of trees. Despite this potential for gaining unique insights from δ2HNEof tree-ring cellulose (δ2HTRC), it has not been widely used compared to other isotope signals, likely due to challenging methodological constraints and interpretations of these isotopic signals. In this chapter, we first summarize hydrogen isotope (2H-) fractionation that occurs between source water and tree rings and review methods (e.g. nitration, equilibration, position-specific applications) and calculations to determine δ2HNE in tree material. Building upon a summary of the current state of knowledge, this chapter also provides an exhaustive synthesis of δ2HTRC papers, applications, and associated data from approximately 180 sites across the globe (paired with modelled precipitation δ2H values and climate data). The data allow us to investigate the hydrological-climatic effects driving δ2HTRC pattern on a global scale, the relationship of hydrogen with oxygen isotopes in the same tree-ring material, as well as the influence of physiological-biochemical effects (e.g., species differences, tree growth) that appear to be more important on local or temporal scales than on a large spatial scales. Thus, when local hydro-climatic influences on source water δ2H can be isolated, δ2HTRC gives novel insights on tree physiological responses to abiotic and biotic stresses. We conclude that the growing constellation of tree-ring metrics, including advancements in 2H-processing (i.e., equilibration techniques allowing rapid determinations of δ2HNE) and further refinements to the understanding of post-photosynthetic 2H-fractionations will together provide many new opportunities to understand past climates and ecophysiology by using δ2H in tree rings.

The analysis of the non-exchangeable hydrogen isotope ratio (δ2Hne) in carbohydrates is mostly limited to the structural component cellulose, while simple high-throughput methods for δ2Hne values of non-structural carbohydrates (NSC) such as sugar and starch do not yet exist. Here, we tested if the hot vapor equilibration method originally developed for cellulose is applicable for NSC, verified by comparison with the traditional nitration method. We set up a detailed analytical protocol and applied the method to plant extracts of leaves from species with different photosynthetic pathways (i.e., C3, C4 and CAM). δ2Hne of commercial sugars and starch from different classes and sources, ranging from −157.8 to +6.4‰, were reproducibly analysed with precision between 0.2‰ and 7.7‰. Mean δ2Hne values of sugar are lowest in C3 (−92.0‰), intermediate in C4 (−32.5‰) and highest in CAM plants (6.0‰), with NSC being 2H-depleted compared to cellulose and sugar being generally more 2H-enriched than starch. Our results suggest that our method can be used in future studies to disentangle 2H-fractionation processes, for improving mechanistic δ2Hne models for leaf and tree-ring cellulose and for further development of δ2Hne in plant carbohydrates as a potential proxy for climate, hydrology, plant metabolism and physiology., Plant, Cell & Environment, 45 (1), ISSN:0140-7791, ISSN:1365-3040

Analysing stable isotope composition of biologic components can be a powerful tool to reconstruct past environmental conditions, physiological responses, and to trace metabolic pathways. The analysis of the carbon-bound non-exchangeable hydrogen isotope ratios (δ2HNE) in carbohydrates can be challenging, partly due to the exchangeability of oxygen-bound hydrogen in the same molecule with those in water or vapour. To eliminate such sample alterations, carbohydrates have been nitrated to substitute exchangeable hydrogen with nitrate ester. However, the nitration of carbohydrates is time consuming, needs high sample amount, has several safety issues, and the nitrated products of short-chained carbohydrates are instable. δ2HNE of sugars derived from living organisms or directly from the environment are thus still limited and not widespread available. Here we optimized recent δ2HNE methods, with the focus on plant-derived non-structural carbohydrates such as starch, sugars, and sugar alcohols. The exchangeable hydrogen is replaced via equilibration with water vapour of a known isotopic composition to calculate δ2HNE. In this presentation, we will explain the new δ2HNE method, discuss precision, accuracy, as well as referencing strategies, and give a first outlook for future applications in plant and environmental sciences.

Steel research international 2100272 (2021). doi:10.1002/srin.202100272, Published by Wiley-VCH-Verl., Weinheim

The analysis of stable carbon and oxygen isotopes in tree-rings is a widely applied tool which allows to retrieve information about past climatic conditions, as well as tree physiological responses to environmental changes. This is based on well-established mechanistic models and firm statistical relationships with climate variables. In contrast, the hydrogen isotopic signature (δ2H) of tree-rings has been reported to be poorly correlated to climate or difficult to explain, and as a consequence, hydrogen isotopes are far less utilized. However, recent plant-physiological experiments have highlighted the role of autotrophic versus heterotrophic processes affecting δ2H values, i.e. use of fresh assimilates versus stored carbohydrates, and have much improved our understanding of the role of post-photosynthetic 2H-fractionation. Using unpublished and literature δ2H data of tree-ring cellulose (δ2HC) of 5 study sites in Europe and Asia, we systematically investigated the relationships between δ2HC and tree-ring width (TRW), which, in contrast to previous research, could now be explained through post-photosynthetic 2H-fractionation. In most cases, these relationships were found to be negative (r2 = 0.23 to 0.51, all P

Calcium phosphate cements (CPCs) have great potential as carriers for controlled release and vectoring of drugs in the skeletal system. However, a lot of work still has to be done in order to obtain reproducible and predictable release kinetics. A particular aspect that adds complexity to these materials is that they cannot be considered as stable matrices, since their microstructure evolves during the setting reaction. The aims of the present work were to analyze the effect of the microstructural evolution of the CPC during the setting reaction on the release kinetics of the antibiotic doxycycline hyclate and to assess the effect of the antibiotic on the microstructural development of the CPC. The incorporation of the drug in the CPC modified the textural and microstructural properties of the cements by acting as a nucleating agent for the heterogeneous precipitation of hydroxyapatite crystals, but did not affect its antibacterial activity. In vitro release experiments were carried out on readily prepared cements (fresh CPCs), and compared to those of pre-set CPCs. No burst release was found in any formulation. A marked difference in release kinetics was found at the initial stages; the evolving microstructure of fresh CPCs led to a two-step release. Initially, when the carrier was merely a suspension of α-TCP particles in water, a faster release was recorded, which rapidly evolved to a zero-order release. In contrast, pre-set CPCs released doxycycline following non-Fickian diffusion. The final release percentage was related to the total porosity and entrance pore size of each biomaterial.