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Climate change not only leads to higher air temperatures but also increases the vapour pressure deficit (VPD) of the air. Understanding the direct effect of VPD on leaf gas exchange is crucial for precise modelling of stomatal functioning. We conducted combined leaf gas exchange and online isotope discrimination measurements on four common European tree species across a VPD range of 0.8-3.6 kPa, while maintaining constant temperatures without soil water limitation. In addition to applying the standard assumption of saturated vapour pressure inside leaves (e i ), we inferred e i from oxygen isotope discrimination of CO 2 and water vapour. e i desaturated progressively with increasing VPD, consistently across species, resulting in an intercellular relative humidity as low as 0.73 ± 0.11 at the highest tested VPD. Assuming saturation of e i overestimated the extent of reductions in stomatal conductance and CO 2 mole fraction inside leaves in response to increasing VPD compared with calculations that accounted for unsaturation. In addition, a significant decrease in mesophyll conductance with increasing VPD only occurred when the unsaturation of e i was considered. We suggest that the possibility of unsaturated e i should not be overlooked in measurements related to leaf gas exchange and in stomatal models, especially at high VPD., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Despite considerable experimental effort, the physiological mechanisms governing temperate tree species' water and carbon dynamics before the onset of the growing period remain poorly understood. We applied 2 H-enriched water during winter dormancy to the soil of four potted European tree species. After 8 weeks of chilling, hydrogen isotopes in stem, twig and bud water were measured six times during 2 consecutive weeks of forcing conditions (Experiment 1). Additionally, we pulse-labelled above-ground plant tissues using 2 H-enriched water vapour and 13 C-enriched CO 2 7 days after exposure to forcing conditions to trace atmospheric water and carbon uptake (Experiment 2). Experiment 1 revealed soil water incorporation into the above-ground organs of all species during the chilling phase and significant species-specific differences in water allocation during the forcing conditions, which we attributed to differences in structural traits. Experiment 2 illustrated water vapour incorporation into all above-ground tissue of all species. However, the incorporation of carbon was found for evergreen saplings only. Our results suggest that temperate trees take up and reallocate soil water and absorb atmospheric water to maintain sufficient above-ground tissue hydration during winter. Therefore, our findings provide new insights into the water allocation dynamics of temperate trees during early spring., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Even though they share many thematical overlaps, plant metabolomics and stable isotope ecology have been rather separate fields mainly due to different mass spectrometry demands. New high-resolution bioanalytical mass spectrometers are now not only offering high-throughput metabolite identification but are also suitable for compound- and intramolecular position-specific isotope analysis in the natural isotope abundance range. In plant metabolomics, label-free metabolic pathway and metabolic flux analysis might become possible when applying this new technology. This is because changes in the commitment of substrates to particular metabolic pathways and the activation or deactivation of others alter enzyme-specific isotope effects. This leads to differences in intramolecular and compound-specific isotope compositions. In plant isotope ecology, position-specific isotope analysis in plant archives informed by metabolic pathway analysis could be used to reconstruct and separate environmental impacts on complex metabolic processes. A technology-driven linkage between the two disciplines could allow us to extract information on environment-metabolism interaction from plant archives such as tree rings but also within ecosystems. This would contribute to a holistic understanding of how plants react to environmental drivers, thus also providing helpful information on the trajectories of the vegetation under the conditions to come., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Measurements of stable isotope ratios in organic compounds are widely used tools for plant ecophysiological studies. However, the complexity of the processes involved in shaping hydrogen isotope values (δ 2 H) in plant carbohydrates has limited its broader application. To investigate the underlying biochemical processes responsible for 2 H fractionation among water, sugars, and cellulose in leaves, we studied the three main CO 2 fixation pathways (C 3 , C 4 , and CAM) and their response to changes in temperature and vapor pressure deficit (VPD). We show significant differences in autotrophic 2 H fractionation (ε A ) from water to sugar among the pathways and their response to changes in air temperature and VPD. The strong 2 H depleting ε A in C 3 plants is likely driven by the photosynthetic H + production within the thylakoids, a reaction that is spatially separated in C 4 and strongly reduced in CAM plants, leading to the absence of 2 H depletion in the latter two types. By contrast, we found that the heterotrophic 2 H-fractionation (ε H ) from sugar to cellulose was very similar among the three pathways and is likely driven by the plant's metabolism, rather than by isotopic exchange with leaf water. Our study offers new insights into the biochemical drivers of 2 H fractionation in plant carbohydrates., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Microcomputed tomography (µCT) is a nondestructive X-ray imaging method used in plant physiology to visualize in situ plant tissues that enables assessments of embolized xylem vessels. Whereas evidence for X-ray-induced cellular damage has been reported, the impact on plant physiological processes such as carbon (C) uptake, transport, and use is unknown. Yet, these damages could be particularly relevant for studies that track embolism and C fluxes over time. We examined the physiological consequences of µCT scanning for xylem embolism over 3 mo by monitoring net photosynthesis (Anet), diameter growth, chlorophyll (Chl) concentration, and foliar nonstructural carbohydrate (NSC) content in 4 deciduous tree species: hedge maple (Acer campestre), ash (Fraxinus excelsior), European hornbeam (Carpinus betulus), and sessile oak (Quercus petraea). C transport from the canopy to the roots was also assessed through 13C labeling. Our results show that monthly X-ray application did not impact foliar Anet, Chl, NSC content, and C transport. Although X-ray effects did not vary between species, the most pronounced impact was observed in sessile oak, marked by stopped growth and stem deformations around the irradiated area. The absence of adverse impacts on plant physiology for all the tested treatments indicates that laboratory-based µCT systems can be used with different beam energy levels and doses without threatening the integrity of plant physiology within the range of tested parameters. However, the impacts of repetitive µCT on the stem radial growth at the irradiated zone leading to deformations in sessile oak might have lasting implications for studies tracking plant embolism in the longer-term., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The hydrogen isotopic composition (δ 2 H) of plant compounds is increasingly used as a hydroclimatic proxy; however, the interpretation of δ 2 H values is hampered by potential coeffecting biochemical and biophysical processes. Here, we studied δ 2 H values of water and carbohydrates in leaves and roots, and of leaf n -alkanes, in two distinct tobacco ( Nicotiana sylvestris ) experiments. Large differences in plant performance and biochemistry resulted from (a) soil fertilization with varying nitrogen (N) species ratios and (b) knockout-induced starch deficiency. We observed a strong 2 H-enrichment in sugars and starch with a decreasing performance induced by increasing NO 3 - /NH 4 + ratios and starch deficiency, as well as from leaves to roots. However, δ 2 H values of cellulose and n -alkanes were less affected. We show that relative concentrations of sugars and starch, interlinked with leaf gas exchange, shape δ 2 H values of carbohydrates. We thus provide insights into drivers of hydrogen isotopic composition of plant compounds and into the mechanistic modeling of plant cellulose δ 2 H values.

The oxygen and hydrogen isotopic composition (δ18O, δ2H) of plant tissues are key tools for the reconstruction of hydrological and plant physiological processes and may therefore be used to disentangle the reasons for tree mortality. However, how both elements respond to soil drought conditions before death has rarely been investigated. To test this, we performed a greenhouse study and determined predisposing fertilization and lethal soil drought effects on δ18O and δ2H values of organic matter in leaves and tree rings of living and dead saplings of five European tree species. For mechanistic insights, we additionally measured isotopic (i.e. δ18O and δ2H values of leaf and twig water), physiological (i.e. leaf water potential and gas-exchange) and metabolic traits (i.e. leaf and stem non-structural carbohydrate concentration, carbon-to-nitrogen ratios). Across all species, lethal soil drought generally caused a homogenous 2H-enrichment in leaf and tree-ring organic matter, but a low and heterogenous δ18O response in the same tissues. Unlike δ18O values, δ2H values of tree-ring organic matter were correlated with those of leaf and twig water and with plant physiological traits across treatments and species. The 2H-enrichment in plant organic matter also went along with a decrease in stem starch concentrations under soil drought compared with well-watered conditions. In contrast, the predisposing fertilization had generally no significant effect on any tested isotopic, physiological and metabolic traits. We propose that the 2H-enrichment in the dead trees is related to (i) the plant water isotopic composition, (ii) metabolic processes shaping leaf non-structural carbohydrates, (iii) the use of carbon reserves for growth and (iv) species-specific physiological adjustments. The homogenous stress imprint on δ2H but not on δ18O suggests that the former could be used as a proxy to reconstruct soil droughts and underlying processes of tree mortality., (© The Author(s) 2024. Published by Oxford University Press.)

Non-structural carbohydrates (NSCs) are building blocks for biomass and fuel metabolic processes. However, it remains unclear how tropical forests mobilize, export, and transport NSCs to cope with extreme droughts. We combined drought manipulation and ecosystem 13CO2 pulse-labeling in an enclosed rainforest at Biosphere 2, assessed changes in NSCs, and traced newly assimilated carbohydrates in plant species with diverse hydraulic traits and canopy positions. We show that drought caused a depletion of leaf starch reserves and slowed export and transport of newly assimilated carbohydrates below ground. Drought effects were more pronounced in conservative canopy trees with limited supply of new photosynthates and relatively constant water status than in those with continual photosynthetic supply and deteriorated water status. We provide experimental evidence that local utilization, export, and transport of newly assimilated carbon are closely coupled with plant water use in canopy trees. We highlight that these processes are critical for understanding and predicting tree resistance and ecosystem fluxes in tropical forest under drought., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The strong covariation of temperature and vapour pressure deficit (VPD) in nature limits our understanding of the direct effects of temperature on leaf gas exchange. Stable isotopes in CO 2 and H 2 O vapour provide mechanistic insight into physiological and biochemical processes during leaf gas exchange. We conducted combined leaf gas exchange and online isotope discrimination measurements on four common European tree species across a leaf temperature range of 5-40°C, while maintaining a constant leaf-to-air VPD (0.8 kPa) without soil water limitation. Above the optimum temperature for photosynthesis (30°C) under the controlled environmental conditions, stomatal conductance (g s ) and net photosynthesis rate (A n ) decoupled across all tested species, with g s increasing but A n decreasing. During this decoupling, mesophyll conductance (cell wall, plasma membrane and chloroplast membrane conductance) consistently and significantly decreased among species; however, this reduction did not lead to reductions in CO 2 concentration at the chloroplast surface and stroma. We question the conventional understanding that diffusional limitations of CO 2 contribute to the reduction in photosynthesis at high temperatures. We suggest that stomata and mesophyll membranes could work strategically to facilitate transpiration cooling and CO 2 supply, thus alleviating heat stress on leaf photosynthetic function, albeit at the cost of reduced water-use efficiency., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

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 δ 13 C values, reflecting lower stomatal restrictions than control trees. Their strong tree-ring δ 18 O and δ 2 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 δ 13 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., (© 2024 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

The oxygen isotope composition (δ 18 O) of tree-ring cellulose is used to evaluate tree physiological responses to climate, but their interpretation is still limited due to the complexity of the isotope fractionation pathways. We assessed the relative contribution of seasonal needle and xylem water δ 18 O variations to the intra-annual tree-ring cellulose δ 18 O signature of larch trees at two sites with contrasting soil water availability in the Swiss Alps. We combined biweekly δ 18 O measurements of soil water, needle water, and twig xylem water with intra-annual δ 18 O measurements of tree-ring cellulose, xylogenesis analysis, and mechanistic and structural equation modeling. Intra-annual cellulose δ 18 O values resembled source water δ 18 O mean levels better than needle water δ 18 O. Large parts of the rings were formed under high proportional exchange with unenriched xylem water (p ex ). Maximum p ex values were achieved in August and imprinted on sections at 50-75% of the ring. High p ex values were associated with periods of high atmospheric evaporative demand (VPD). While VPD governed needle water δ 18 O variability, we estimated a limited Péclet effect at both sites. Due to a variable p ex , source water has a strong influence over large parts of the intra-annual tree-ring cellulose δ 18 O variations, potentially masking signals coming from needle-level processes., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Oxygen and hydrogen isotopes of cellulose in plant biology are commonly used to infer environmental conditions, often from time series measurements of tree rings. However, the covariation (or the lack thereof) between δ 18 O and δ 2 H in plant cellulose is still poorly understood. We compared plant water, and leaf and branch cellulose from dominant tree species across an aridity gradient in Northern Australia, to examine how δ 18 O and δ 2 H relate to each other and to mean annual precipitation (MAP). We identified a decline in covariation from xylem to leaf water, and onwards from leaf to branch wood cellulose. Covariation in leaf water isotopic enrichment (Δ) was partially preserved in leaf cellulose but not branch wood cellulose. Furthermore, whilst δ 2 H was well-correlated between leaf and branch, there was an offset in δ 18 O between organs that increased with decreasing MAP. Our findings strongly suggest that postphotosynthetic isotope exchange with water is more apparent for oxygen isotopes, whereas variable kinetic and nonequilibrium isotope effects add complexity to interpreting metabolic-induced δ 2 H patterns. Varying oxygen isotope exchange in wood and leaf cellulose must be accounted for when δ 18 O is used to reconstruct climatic scenarios. Conversely, comparing δ 2 H and δ 18 O patterns may reveal environmentally induced shifts in metabolism., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

The world is on the verge of a new industrial revolution, and language models are poised to play a pivotal role in this transformative era. Their ability to offer intelligent insights and forecasts has made them a valuable asset for businesses seeking a competitive advantage. The chemical industry, in particular, can benefit significantly from harnessing their power. Since 2016 already, language models have been applied to tasks such as predicting reaction outcomes or retrosynthetic routes. While such models have demonstrated impressive abilities, the lack of publicly available data sets with universal coverage is often the limiting factor for achieving even higher accuracies. This makes it imperative for organizations to incorporate proprietary data sets into their model training processes to improve their performance. So far, however, these data sets frequently remain untapped as there are no established criteria for model customization. In this work, we report a successful methodology for retraining language models on reaction outcome prediction and single-step retrosynthesis tasks, using proprietary, nonpublic data sets. We report a considerable boost in accuracy by combining patent and proprietary data in a multidomain learning formulation. This exercise, inspired by a real-world use case, enables us to formulate guidelines that can be adopted in different corporate settings to customize chemical language models easily., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors and Syngenta. Published by American Chemical Society.)

The combined study of carbon (C) and oxygen (O) isotopes in plant organic matter has emerged as a powerful tool for understanding plant functional responses to environmental change. The approach relies on established relationships between leaf gas exchange and isotopic fractionation to derive a series of model scenarios that can be used to infer changes in photosynthetic assimilation and stomatal conductance driven by changes in environmental parameters (CO 2 , water availability, air humidity, temperature, nutrients). We review the mechanistic basis for a conceptual model, in light of recently published research, and discuss where isotopic observations do not match our current understanding of plant physiological response to the environment. We demonstrate that (1) the model was applied successfully in many, but not all studies; (2) although originally conceived for leaf isotopes, the model has been applied extensively to tree-ring isotopes in the context of tree physiology and dendrochronology. Where isotopic observations deviate from physiologically plausible conclusions, this mismatch between gas exchange and isotope response provides valuable insights into underlying physiological processes. Overall, we found that isotope responses can be grouped into situations of increasing resource limitation versus higher resource availability. The dual-isotope model helps to interpret plant responses to a multitude of environmental factors., (© 2023 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Carbon isotope composition of tree-ring (δ 13 C Ring ) is a commonly used proxy for environmental change and ecophysiology. δ 13 C Ring reconstructions are based on a solid knowledge of isotope fractionations during formation of primary photosynthates (δ 13 C P ), such as sucrose. However, δ 13 C Ring is not merely a record of δ 13 C P . Isotope fractionation processes, which are not yet fully understood, modify δ 13 C P during sucrose transport. We traced, how the environmental intra-seasonal δ 13 C P signal changes from leaves to phloem, tree-ring and roots, for 7 year old Pinus sylvestris, using δ 13 C analysis of individual carbohydrates, δ 13 C Ring laser ablation, leaf gas exchange and enzyme activity measurements. The intra-seasonal δ 13 C P dynamics was clearly reflected by δ 13 C Ring , suggesting negligible impact of reserve use on δ 13 C Ring . However, δ 13 C P became increasingly 13 C-enriched during down-stem transport, probably due to post-photosynthetic fractionations such as sink organ catabolism. In contrast, δ 13 C of water-soluble carbohydrates, analysed for the same extracts, did not reflect the same isotope dynamics and fractionations as δ 13 C P , but recorded intra-seasonal δ 13 C P variability. The impact of environmental signals on δ 13 C Ring , and the 0.5 and 1.7‰ depletion in photosynthates compared ring organic matter and tree-ring cellulose, respectively, are useful pieces of information for studies exploiting δ 13 C Ring ., (© 2023 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Recent methodological advancements in determining the nonexchangeable hydrogen isotopic composition (δ 2 H ne ) of plant carbohydrates make it possible to disentangle the drivers of hydrogen isotope ( 2 H) fractionation processes in plants. Here, we investigated the influence of phylogeny on the δ 2 H ne 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. 2 H fractionation in plant carbohydrates followed distinct phylogenetic patterns, with phylogeny reflected more in the δ 2 H ne of leaf sugars than in that of twig xylem cellulose. Phylogeny had no detectable influence on the δ 2 H ne of twig or leaf water, showing that biochemistry, not isotopic differences in plant water, caused the observed phylogenetic pattern in carbohydrates. Angiosperms were more 2 H-enriched than gymnosperms, but substantial δ 2 H ne variations also occurred at the order, family, and species levels within both clades. Differences in the strength of the phylogenetic signals in δ 2 H ne 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 2 H fractionation models for plant carbohydrates and have important consequences for dendrochronological and ecophysiological studies., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Stable isotope ratio analysis of tree rings has been widely and successfully applied in recent decades for climatic and environmental reconstructions. These studies were mostly conducted at an annual resolution, considering one measurement per tree ring, often focusing on latewood. However, much more information could be retrieved with high-resolution intra-annual isotope studies, based on the fact that the wood cells and the corresponding organic matter are continuously laid down during the growing season. Such studies are still relatively rare, but have a unique potential for reconstructing seasonal climate variations or short-term changes in physiological plant properties, like water-use efficiency. The reason for this research gap is mostly technical, as on the one hand sub-annual, manual splitting of rings is very tedious, while on the other hand automated laser ablation for high-resolution analyses is not yet well established and available. Here, we give an update on the current status of laser ablation research for analysis of the carbon isotope ratio (δ13C) of wood, describe an easy-to-use laser ablation system, its operation and discuss practical issues related to tree core preparation, including cellulose extraction. The results show that routine analysis with up to 100 laser shot-derived δ13C-values daily and good precision and accuracy (ca. 0.1‰) comparable to conventional combustion in an elemental analyzer are possible. Measurements on resin-extracted wood is recommended as most efficient, but laser ablation is also possible on cellulose extracted wood pieces. Considering the straightforward sample preparation, the technique is therefore ripe for wide-spread application. With this work, we hope to stimulate future progress in the promising field of high-resolution environmental reconstruction using laser ablation., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Recent experiments have underlined the potential of δ2H in tree-ring cellulose as a physiological indicator of shifts in autotrophic versus heterotrophic processes (i.e., the use of fresh versus stored non-structural carbohydrates). However, the impact of these processes has not yet been quantified under natural conditions. Defoliator outbreaks disrupt tree functioning and carbon assimilation, stimulating remobilization, therefore providing a unique opportunity to improve our understanding of changes in δ2H. By exploring a 700-year tree-ring isotope chronology from Switzerland, we assessed the impact of 79 larch budmoth (LBM, Zeiraphera griseana [Hübner]) outbreaks on the growth of its host tree species, Larix decidua [Mill]. The LBM outbreaks significantly altered the tree-ring isotopic signature, creating a 2H-enrichment and an 18O- and 13C-depletion. Changes in tree physiological functioning in outbreak years are shown by the decoupling of δ2H and δ18O (O-H relationship), in contrast to the positive correlation in non-outbreak years. Across the centuries, 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. We conclude that the combination of these isotopic parameters can serve as a metric for assessing changes in physiological mechanisms over time., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Increasing frequencies of heatwaves combined with simultaneous drought stress in Europe threaten the ecosystem water and carbon budgets of alpine grasslands. Dew as an additional water source can promote ecosystem carbon assimilation. It is known that grassland ecosystems keep high evapotranspiration as long as soil water is available. However, it is rarely being investigated whether dew can mitigate the impact of such extreme climatic events on grassland ecosystem carbon and water exchange. Here we use stable isotopes in meteoric waters and leaf sugars, eddy covariance fluxes for H 2 O vapor and CO 2 , in combination with meteorological and plant physiological measurements, to investigate the combined effect of dew and heat-drought stress on plant water status and net ecosystem production (NEP) in an alpine grassland (2000 m elevation) during the June 2019 European heatwave. Before the heatwave, enhanced NEP in the early morning hours can be attributed to leaf wetting by dew. However, dew benefits on NEP were cancelled out by the heatwave, due to the minor contribution of dew in leaf water. Heat-induced reduction in NEP was intensified by the combined effect of drought stress. The recovery of NEP after the peak of the heatwave could be linked to the refilling of plant tissues during nighttime. Among-genera differences of plant water status affected by dew and heat-drought stress can be attributed to differences in their foliar dew water uptake, and their reliance on soil moisture or the impact of the atmospheric evaporative demand. Our results indicate that dew influence on alpine grassland ecosystems varies according to the environmental stress and plant physiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Eugster, Riedl, Lehmann, Aemisegger and Buchmann.)

Introduction: European mistletoe, Viscum album L., is a hemiparasite that can infect various tree species, yet our understanding of its physiological interactions with host species is limited., Methods: Nine mistletoe-host pairs (i.e. V. album ssp. album growing on nine different broadleaf tree species) under different growth conditions in central Switzerland were selected to examine the carbon, water and nutrient relationships between mistletoe and its hosts. We measured leaf morphological traits, isotopic compositions (δ13C and δ15N), concentrations of non-structural carbohydrates (NSC) and specific compounds (i.e. mobile sugars and starch), and macronutrients (i.e. N, P, K, Ca, Mg, S) in leaf and xylem tissues of both mistletoe and its hosts., Results and Discussion: There were only non-significant relationships between NSC concentrations in mistletoe and in its host species across the nine mistletoe-host pairs, suggesting the carbon condition of V. album ssp. album is determined by both the heterotrophic carbon transfer and self-photosynthetic capacity among different mistletoe-host pairs. However, mistletoe leaf morphological traits (single leaf area and mass, and leaf mass per unit leaf area) did not change across the nine mistletoe-host pairs, and mistletoe leaf δ13C, water content and macronutrient concentrations were linearly correlated with those in the host leaves. Macronutrients showed accumulations in mistletoe across the nine pairs. Further, tissue N concentrations were significantly higher in mistletoe grown on N-fixing hosts than on non-N-fixing hosts. Finally, leaf N:P in mistletoe was significantly correlated with the ratio in the host across the nine mistletoe-host pairs. Overall, our results indicate strong relationships between mistletoe and its hosts for water- and nutrient-related traits, but not for carbon-related traits, which demonstrates that V. album ssp. album can adjust its physiology to survive on different deciduous tree species hosts and under different site conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Bose, Lehmann, Rigling, Gessler, Yu and Li.)

Many carbon-related physiological questions in plants such as carbon (C) limitation or starvation have not yet been resolved thoroughly due to the lack of suitable experimental methodology. As a first step towards resolving these problems, we conducted infusion experiments with bonsai trees ( Ficus microcarpa ) and young maple trees ( Acer pseudoplatanus ) in greenhouse, and with adult Scots pine trees ( Pinus sylvestris ) in the field, that were "fed" with 13 C-labelled glucose either through the phloem or the xylem. We then traced the 13 C-signal in plant organic matter and respiration to test whether trees can take up and metabolize exogenous sugars infused. Ten weeks after infusion started, xylem but not phloem infusion significantly increased the δ 13 C values in both aboveground and belowground tissues of the bonsai trees in the greenhouse, whereas xylem infusion significantly increased xylem δ 13 C values and phloem infusion significantly increased phloem δ 13 C values of the adult pines in the field experiment, compared to the corresponding controls. The respiration measurement experiment with young maple trees showed significantly increased δ 13 C-values in shoot respired CO 2 at the time of four weeks after xylem infusion started. Our results clearly indicate that trees do translocate and metabolize exogenous sugars infused, and because the phloem layer is too thin, and thus xylem infusion can be better operated than phloem infusion. This tree infusion method developed here opens up new avenues and has great potential to be used for research on the whole plant C balance and its regulation in response to environmental factors and extreme stress conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Yang, Saurer, Schaub, Gessler, Lehmann, Rigling, Walser, Stierli, Hajjar, Christen and Li.)

Sucrose has a unique role in recording environmental and physiological signals during photosynthesis in its carbon isotope composition (δ13C) and transport of the signal to tree rings. Yet, instead of sucrose, total organic matter (TOM) or water-soluble carbohydrates (WSC) are typically analysed in studies that follow δ13C signals within trees. To study how the choice of organic material may bias the interpretation of δ13C records, we used mature field-grown Scots pine (Pinus sylvestris) to compare for the first time δ13C of different leaf carbon pools with δ13C of assimilates estimated by a chamber-Picarro system (δ13CA_Picarro), and a photosynthetic discrimination model (δ13CA_model). Compared with sucrose, the other tested carbon pools, such as TOM and WSC, poorly recorded the seasonal trends or absolute values of δ13CA_Picarro and δ13CA_model. Consequently, in comparison with the other carbon pools, sucrose δ13C was superior for reconstructing changes in intrinsic water use efficiency (iWUE), agreeing in both absolute values and intra-seasonal variations with iWUE estimated from gas exchange. Thus, deriving iWUE and environmental signals from δ13C of bulk organic matter can lead to misinterpretation. Our findings underscore the advantage of using sucrose δ13C to understand plant physiological responses in depth., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

After drought events, tree recovery depends on sufficient carbon (C) allocation to the sink organs. The present study aimed to elucidate dynamics of tree-level C sink activity and allocation of recent photoassimilates (C new ) and stored C in c. 70-year-old Norway spruce (Picea abies) trees during a 4-week period after drought release. We conducted a continuous, whole-tree 13 C labeling in parallel with controlled watering after 5 years of experimental summer drought. The fate of C new to growth and CO 2 efflux was tracked along branches, stems, coarse- and fine roots, ectomycorrhizae and root exudates to soil CO 2 efflux after drought release. Compared with control trees, drought recovering trees showed an overall 6% lower C sink activity and 19% less allocation of C new to aboveground sinks, indicating a low priority for aboveground sinks during recovery. In contrast, fine-root growth in recovering trees was seven times greater than that of controls. However, only half of the C used for new fine-root growth was comprised of C new while the other half was supplied by stored C. For drought recovery of mature spruce trees, in addition to C new , stored C appears to be critical for the regeneration of the fine-root system and the associated water uptake capacity., (© 2022 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

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., (© 2022 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

The impact of climate extremes on forest ecosystems is poorly understood but important for predicting carbon and water cycle feedbacks to climate. Some knowledge gaps still remain regarding how drought-related adjustments in intra-annual tree-ring characteristics directly impact tree carbon and water use. In this study we quantified the impact of an extreme summer drought on the water-use efficiency and carbon sequestration of four mature Norway spruce trees. We used detailed observations of wood formation (xylogenesis) and intra-annual tree-ring properties (quantitative wood anatomy and stable carbon isotopes) combined with physiological water-stress monitoring. During 41 d of tree water deficit, we observed an enrichment in 13 C but a reduction in cell enlargement and wall-thickening processes, which impacted the anatomical characteristics. These adjustments diminished carbon sequestration by 67% despite an 11% increase in water-use efficiency during drought. However, with the resumption of a positive hydric state in the stem, we observed a fast recovery of cell formation rates based on the accumulated assimilates produced during drought. Our findings enhance our understanding of carbon and water fluxes between the atmosphere and forest ecosystems, providing observational evidence on the tree intra-annual carbon sequestration and water-use efficiency dynamics to improve future generations of vegetation models., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Experimental approaches to isolate drivers of variation in the carbon-bound hydrogen isotope composition (δ 2 H) of plant cellulose are rare and current models are limited in their application. This is in part due to a lack in understanding of how 2 H-fractionations in carbohydrates differ between species. We analysed, for the first time, the δ 2 H of leaf sucrose along with the δ 2 H and δ 18 O of leaf cellulose and leaf and xylem water across seven herbaceous species and a starchless mutant of tobacco. The δ 2 H of sucrose explained 66% of the δ 2 H variation in cellulose (R 2  = 0.66), which was associated with species differences in the 2 H enrichment of sucrose above leaf water ( ε sucrose : -126% to -192‰) rather than by variation in leaf water δ 2 H itself. ε sucrose was positively related to dark respiration (R 2  = 0.27), and isotopic exchange of hydrogen in sugars was positively related to the turnover time of carbohydrates (R 2  = 0.38), but only when ε sucrose was fixed to the literature accepted value of - 171 ‰. No relation was found between isotopic exchange of hydrogen and oxygen, suggesting large differences in the processes shaping post-photosynthetic fractionation between elements. Our results strongly advocate that for robust applications of the leaf cellulose hydrogen isotope model, parameterization utilizing δ 2 H of sugars is needed., (© 2022 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Understanding δ18O and δ2H values of agricultural products like fruit is of particular scientific interest in plant physiology, ecology, and forensic studies. Applications of mechanistic stable isotope models to predict δ18O and δ2H values of water and organic compounds in fruit, however, are hindered by a lack of empirical parameterizations and validations. We addressed this lack of data by experimentally evaluating model parameter values required to model δ18O and δ2H values of water and organic compounds in berries and leaves from strawberry and raspberry plants grown at different relative humidities. Our study revealed substantial differences between leaf and berry isotope values, consistent across the different relative humidity treatments. We demonstrated that existing isotope models can reproduce water and organic δ18O and δ2H values for leaves and berries. Yet, these simulations require organ-specific model parameterization to accurately predict δ18O and δ2H values of leaf and berry tissue and water pools. We quantified these organ-specific model parameters for both species and relative humidity conditions. Depending on the required model accuracy, species- and environment-specific model parameters may be justified. The parameter values determined in this study thus facilitate applications of stable isotope models where understanding δ18O and δ2H values of fruit is of scientific interest., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Pine mistletoe is a hemiparasitic shrub that can produce its own photosynthates. There is a lack of knowledge about the interaction of mistletoe and host under varying environmental condition that might influence carbon gain and allocation. In a 13 C-pulse labeling experiment with mature Pinus sylvestris (pine) infected by mistletoes grown in naturally dry or irrigated conditions, (1) mistletoe clusters were shielded from 13 CO 2 added, and (2) mistletoes or host needles were removed to manipulate the local assimilate and water availability. No 13 C signal was found in shielded mistletoes, indicating no carbon transfer from the host to the mistletoe. When the pine needles were removed from girdled branches, no 13 C signal was found in the host tissues, implying no carbon transfer from mistletoe to the host. However, mistletoes on needle-removed pine trees accumulated more labeled assimilates and had higher non-structural carbohydrate (NSC) concentrations only under naturally dry conditions but not in irrigated plots. Our results suggest that mistletoes show full carbon autonomy, as they neither receive carbon from nor provide carbon resource to the host trees. Moreover, the high assimilation capacity of mistletoes seems to be constrained by the host water use under dry conditions, suggesting that drought stress is not only negatively impacting trees but also mistletoes. Therefore, we conclude that the hemiparasites live on their own in terms of carbon gain which, however, depends on the water provided by the host tree., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Lehmann, Rigling, Gessler, Saurer, Du and Li.)

This is the first Europe-wide comprehensive assessment of the climatological and physiological information recorded by hydrogen isotope ratios in tree-ring cellulose (δ 2 H c ) based on a unique collection of annually resolved 100-year tree-ring records of two genera (Pinus and Quercus) from 17 sites (36°N to 68°N). We observed that the high-frequency climate signals in the δ 2 H c chronologies were weaker than those recorded in carbon (δ 13 C c ) and oxygen isotope signals (δ 18 O c ) but similar to the tree-ring width ones (TRW). The δ 2 H c climate signal strength varied across the continent and was stronger and more consistent for Pinus than for Quercus. For both genera, years with extremely dry summer conditions caused a significant 2 H-enrichment in tree-ring cellulose. The δ 2 H c inter-annual variability was strongly site-specific, as a result of the imprinting of climate and hydrology, but also physiological mechanisms and tree growth. To differentiate between environmental and physiological signals in δ 2 H c , we investigated its relationships with δ 18 O c and TRW. We found significant negative relationships between δ 2 H c and TRW (7 sites), and positive ones between δ 2 H c and δ 18 O c (10 sites). The strength of these relationships was nonlinearly related to temperature and precipitation. Mechanistic δ 2 H c models performed well for both genera at continental scale simulating average values, but they failed on capturing year-to-year δ 2 H c variations. Our results suggest that the information recorded by δ 2 H c is significantly different from that of δ 18 O c , and has a stronger physiological component independent from climate, possibly related to the use of carbohydrate reserves for growth. Advancements in the understanding of 2 H-fractionations and their relationships with climate, physiology, and species-specific traits are needed to improve the modelling and interpretation accuracy of δ 2 H c . Such advancements could lead to new insights into trees' carbon allocation mechanisms, and responses to abiotic and biotic stress conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Under ongoing global climate change, drought periods are predicted to increase in frequency and intensity in the future. Under these circumstances, it is crucial for tree's survival to recover their restricted functionalities quickly after drought release. To elucidate the recovery of carbon (C) transport rates in c. 70-year-old Norway spruce (Picea abies [L.] KARST.) after 5 years of recurrent summer droughts, we conducted a continuous whole-tree 13 C labeling experiment in parallel with watering. We determined the arrival time of current photoassimilates in major C sinks by tracing the 13 C label in stem and soil CO 2 efflux, and tips of living fine roots. In the first week after watering, aboveground C transport rates (CTR) from crown to trunk base were still 50% lower in previously drought-stressed trees (0.16 ± 0.01 m h -1 ) compared to controls (0.30 ± 0.06 m h -1 ). Conversely, CTR below ground, that is, from the trunk base to soil CO 2 efflux were already similar between treatments (c. 0.03 m h -1 ). Two weeks after watering, aboveground C transport of previously drought-stressed trees recovered to the level of the controls. Furthermore, regrowth of water-absorbing fine roots upon watering was supported by faster incorporation of 13 C label in previously drought-stressed (within 12 ± 10 h upon arrival at trunk base) compared to control trees (73 ± 10 h). Thus, the whole-tree C transport system from the crown to soil CO 2 efflux fully recovered within 2 weeks after drought release, and hence showed high resilience to recurrent summer droughts in mature Norway spruce forests. This high resilience of the C transport system is an important prerequisite for the recovery of other tree functionalities and productivity., (© 2021 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

Heatwaves combined with drought affect tree functioning with as yet undetermined legacy effects on carbon (C) and nitrogen (N) allocation. We continuously monitored shoot and root gas exchange, δ 13 CO 2 of respiration and stem growth in well-watered and drought-treated Pinus sylvestris (Scots pine) seedlings exposed to increasing daytime temperatures (max. 42°C) and evaporative demand. Following stress release, we used 13 CO 2 canopy pulse-labeling, supplemented by soil-applied 15 N, to determine allocation to plant compartments, respiration and soil microbial biomass (SMB) over 2.5 wk. Previously heat-treated seedlings rapidly translocated 13 C along the long-distance transport path, to root respiration (R root ; 7.1 h) and SMB (3 d). Furthermore, 13 C accumulated in branch cellulose, suggesting secondary growth enhancement. However, in recovering drought-heat seedlings, the mean residence time of 13 C in needles increased, whereas C translocation to R root was delayed (13.8 h) and 13 C incorporated into starch rather than cellulose. Concurrently, we observed stress-induced low N uptake and aboveground allocation. C and N allocation during early recovery were affected by stress type and impact. Although C uptake increased quickly in both treatments, drought-heat in combination reduced the above-belowground coupling and starch accumulated in leaves at the expense of growth. Accordingly, C allocation during recovery depends on phloem translocation capacity., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

The analysis of the non-exchangeable hydrogen isotope ratio (δ 2 H ne ) in carbohydrates is mostly limited to the structural component cellulose, while simple high-throughput methods for δ 2 H ne 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., C 3 , C 4 and CAM). δ 2 H ne 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 δ 2 H ne values of sugar are lowest in C 3 (-92.0‰), intermediate in C 4 (-32.5‰) and highest in CAM plants (6.0‰), with NSC being 2 H-depleted compared to cellulose and sugar being generally more 2 H-enriched than starch. Our results suggest that our method can be used in future studies to disentangle 2 H-fractionation processes, for improving mechanistic δ 2 H ne models for leaf and tree-ring cellulose and for further development of δ 2 H ne in plant carbohydrates as a potential proxy for climate, hydrology, plant metabolism and physiology., (© 2021 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Severe droughts endanger ecosystem functioning worldwide. We investigated how drought affects carbon and water fluxes as well as soil-plant-atmosphere interactions by tracing 13 CO 2 and deep water 2 H 2 O label pulses and volatile organic compounds (VOCs) in an enclosed experimental rainforest. Ecosystem dynamics were driven by different plant functional group responses to drought. Drought-sensitive canopy trees dominated total fluxes but also exhibited the strongest response to topsoil drying. Although all canopy-forming trees had access to deep water, these reserves were spared until late in the drought. Belowground carbon transport was slowed, yet allocation of fresh carbon to VOCs remained high. Atmospheric VOC composition reflected increasing stress responses and dynamic soil-plant-atmosphere interactions, potentially affecting atmospheric chemistry and climate feedbacks. These interactions and distinct functional group strategies thus modulate drought impacts and ecosystem susceptibility to climate change.

The capacity of trees to release water from storage compartments into the transpiration stream can mitigate damage to hydraulic functioning. However, the location of these 'transient' water sources and also the pathways of water movement other than vertical through tree stems still remain poorly understood. We conducted an experiment on two tree species in a common garden in eastern Australia that naturally grow in regions of high (Eucalyptus tereticornis, 'Red Gum') and low (Eucalyptus sideroxylon, 'Ironbark') annual precipitation rates. Deuterium-enriched water (1350% label strength) was directly introduced into the transpiration stream of three trees per species for four consecutive days. Subsequently, the trees were felled, woody tissue samples were collected from different heights and azimuthal positions of the stems, and stable isotope ratios were determined on the water extracted from all samples. The presence/absence of the tracer along the radial and vertical stem axes in combination with xylem hydraulic properties inferred from sapflow, leaf and stem water potentials, wood moisture contents and anatomical sapwood characteristics elucidated species-specific patterns of short-term stem water storage and movement. The distribution of water isotopes at natural abundance among woody tissues indicated systematic differences with highest values of sapwood water and lower values in inner bark and heartwood. Presence of tracer in water of the inner bark highlighted the importance of this tissue as capacitor. Although injected at the northern side of stems, tracer was also discovered at the southern side, providing empirical evidence for circumferential flow in sapwood, particularly of Ironbark. Greater vertical water transport in Red Gum compared with more radial and circumferential water transport in Ironbark were associated with species-specific sapwood anatomy. Our study highlights the value of combining information from stable isotope tracers and wood anatomy to investigate patterns of water transport and storage of tall trees in situ., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Canary Islands, an archipelago east of Morocco's Atlantic coast, present steep altitudinal gradients covering various climatic zones from hot deserts to subalpine Mediterranean, passing through fog-influenced cloud forests. Unlike the majority of the Canarian flora, Pinus canariensis C. Sm. ex DC. in Buch grow along most of these gradients, allowing the study of plant functioning in contrasting ecosystems. Here we assess the water sources (precipitation, fog) of P. canariensis and its physiological behavior in its different natural environments. We analyzed carbon and oxygen isotope ratios of water and organics from atmosphere, soil and different plant organs and tissues (including 10-year annual time series of tree-ring cellulose) of six sites from 480 to 1990 m above sea level on the Canary Island La Palma. We found a decreasing δ18O trend in source water that was overridden by an increasing δ18O trend in needle water, leaf assimilates and tree-ring cellulose with increasing altitude, suggesting site-specific tree physiological responses to relative humidity. Fog-influenced and fog-free sites showed similar δ13C values, suggesting photosynthetic activity to be limited by stomatal closure and irradiance at certain periods. In addition, we observed an 18O-depletion (fog-free and timberline sites) and 13C-depletion (fog-influenced and fog-free sites) in latewood compared with earlywood caused by seasonal differences in: (i) water uptake (i.e., deeper ground water during summer drought, fog water frequency and interception) and (ii) meteorological conditions (stem radial growth and latewood δ18O correlated with winter precipitation). In addition, we found evidence for foliar water uptake and strong isotopic gradients along the pine needle axis in water and assimilates. These gradients are likely the reason for an unexpected underestimation of pine needle water δ18O when applying standard leaf water δ18O models. Our results indicate that soil water availability and air humidity conditions are the main drivers of the physiological behavior of pine along the Canary Island's altitudinal gradients., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Drought alters allocation patterns of carbon (C) and nutrients in trees and eventually impairs tree functioning. Elevated soil nutrient availability might alter the response of trees to drought. We hypothesize that increased soil nutrient availability stimulates root metabolism and C allocation to belowground tissues under drought stress. To test this hypothesis, we subjected 3-year-old Pinus sylvestris L. saplings in open-top chambers during two subsequent years to drought using three different water treatments (100, 20 and 0% plant available water in the soil) and two soil nutrient regimes (ambient and nitrogen-phosphorus-potassium (N-P-K) fertilization corresponding to 5 g N m-2 year-1) and released drought thereafter. We conducted a 15N and 13C labeling experiment during the peak of the first-year drought by injecting 15N labeled fertilizer in the soil and exposing the tree canopies to 13C labeled CO2. The abundance of the N and C isotopes in the roots, stem and needles was assessed during the following year. Carbon uptake was slightly lower in drought-stressed trees, and extreme drought inhibited largely the N uptake and transport. Carbon allocation to belowground tissues was decreased under drought, but not in combination with fertilization. Our results indicate a potential positive feedback loop, where fertilization improved the metabolism and functioning of the roots, stimulating C allocation to belowground tissues. This way, soil nutrients compensated for drought-induced loss of root functioning, mitigating drought stress of trees., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

The carbon isotopic composition (δ 13 C) of foliage is often used as proxy for plant performance. However, the effect of N O 3 - vs. N H 4 + supply on δ 13 C of leaf metabolites and respired CO 2 is largely unknown. We supplied tobacco plants with a gradient of N O 3 - to N H 4 + concentration ratios and determined gas exchange variables, concentrations and δ 13 C of tricarboxylic acid (TCA) cycle intermediates, δ 13 C of dark-respired CO 2 , and activities of key enzymes nitrate reductase, malic enzyme and phosphoenolpyruvate carboxylase. Net assimilation rate, dry biomass and concentrations of organic acids and starch decreased along the gradient. In contrast, respiration rates, concentrations of intercellular CO 2 , soluble sugars and amino acids increased. As N O 3 - decreased, activities of all measured enzymes decreased. δ 13 C of CO 2 and organic acids closely co-varied and were more positive under N O 3 - supply, suggesting organic acids as potential substrates for respiration. Together with estimates of intra-molecular 13 C enrichment in malate, we conclude that a change in the anaplerotic reaction of the TCA cycle possibly contributes to 13 C enrichment in organic acids and respired CO 2 under N O 3 - supply. Thus, the effect of N O 3 - vs. N H 4 + on δ 13 C is highly relevant, particularly if δ 13 C of leaf metabolites or respiration is used as proxy for plant performance.

The carbon and oxygen isotopic composition of water and assimilates in plants reveals valuable information on plant responses to climatic conditions. Yet, the carbon and oxygen uptake, incorporation and allocation processes determining isotopic compositions are not fully understood. We carried out a dual-isotope labeling experiment at high humidity with 18O-enriched water (H218O) and 13C-enriched CO2 (13CO2) with attached Scots pine (Pinus sylvestris L.) branches and detached twigs of hemiparasitic mistletoes (Viscum album ssp. austriacum) in a naturally dry coniferous forest, where also a long-term irrigation takes place. After 4 h of label exposure, we sampled previous- and recent-year leaves, twig phloem and twig xylem over 192 h for the analysis of isotope ratios in water and assimilates. For both species, the uptake into leaf water and the incorporation of the 18O-label into leaf assimilates was not influenced by soil moisture, while the 13C-label incorporation into assimilates was significantly higher under irrigation compared with control dry conditions. Species-specific differences in leaf morphology or needle age did not affect 18O-label uptake into leaf water, but the incorporation of both tracers into assimilates was two times lower in mistletoe than in pine. The 18O-label allocation in water from pine needles to twig tissues was two times higher for phloem than for xylem under both soil moisture conditions. In contrast, the allocation of both tracers in pine assimilates were similar and not affected by soil moisture, twig tissue or needle age. Soil moisture effects on 13C-label but not on 18O-label incorporation into assimilates can be explained by the stomatal responses at high humidity, non-stomatal pathways for water and isotope exchange reactions. Our results suggest that non-photosynthetic 18O-incorporation processes may have masked prevalent photosynthetic processes. Thus, isotopic variation in leaf water could also be imprinted on assimilates when photosynthetic assimilation rates are low., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Insect defoliation impacts forest productivity worldwide, highlighting the relevance of plant-insect interactions. The larch budmoth (Zeiraphera griseana Hübner) is one of the most extensively studied defoliators, where numerous tree ring-based analyses on its host (Larix decidua Mill.) have aided in identifying outbreak dynamics over the past millennia. Yet, outbreaks have been widely absent after the early 1980s, and little is known about the in situ tree physiological responses and the allocation of carbon resources during and after defoliation. In summer 2018, we tracked an ongoing larch budmoth outbreak in a well-studied larch forest in the Swiss Alps. We performed biweekly monitoring on an affected and unaffected site using a unique combination of xylogenesis observations, measurements of non-structural carbohydrates, isotopic analysis of needle assimilates and ground-based and remote-sensed leaf trait observations. The budmoth induced a defoliation that lasted 40 days and could be detected by satellite observations. Soluble sugars significantly decreased in needles and stem phloem of the defoliated trees, while starch levels remained stable in the stem and root xylem compared to the control. Carbon and oxygen isotope ratios in needle assimilates indicated that neither photosynthetic assimilation rates nor stomatal conductance was different between sites before, during and after the outbreak. Defoliated trees ceased cell wall thickening 17 days earlier than unaffected trees, showing the earliest halt of ring formation recorded from 2007 untill 2013 and causing significant thinner cell walls, particularly in the latewood. No significant differences were found for cell enlargement rates and ring width. Our study revealed that an outbreak causes a downregulation of cell wall thickening first, while no starch is mobilized or leaf physiology is adjusted to compensate for the reduced carbon source due to defoliation. Our observations suggest that affected larch trees prioritize leaf recovery and carbon storage over wood biomass development., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rationale: The oxygen isotopic composition (here shown as the δ 18 O value) of soluble sugars in leaves and phloem tissue holds valuable information about plant functions in response to climatic changes. However, δ 18 O analysis of sugars is prone to error, and thoroughly tested methods are lacking., Methods: We performed three experiments to test if sample preparation modifies the δ 18 O values of sugars. In experiment 1, we tested the effects of oven-drying versus freeze-drying, whereas in experiment 2 we focused on the extraction and purification of leaf sugars. In experiment 3, we investigated the exudation and purification of twig phloem sugars as a function of exudation time and different ethylenediaminetetraacetic acid (EDTA) exudation media., Results: Freeze-drying produced more consistent δ 18 O values than oven-drying for sucrose but not for phloem sugars. The extraction and purification of leaf sugars can be performed without a significant modification of their δ 18 O values; yet the purified leaf and phloem sugars possessed higher δ 18 O values than the fraction of water-soluble compounds. Moreover, the exudation time significantly modulated the δ 18 O values of phloem sugars, which is probably related to changes in the sugar composition. The addition of EDTA did not improve the determination of the δ 18 O values of phloem sugars., Conclusions: We show that the sample preparation of plant sugars for the reliable determination of δ 18 O values requires a strict protocol, which is described in this paper. For phloem sugar, we recommend a maximum exudation time of 1 h to reduce the degradation of sucrose and minimise oxygen isotope exchange reactions between the resulting hexoses and water., (© 2020 John Wiley & Sons, Ltd.)

Drought alters carbon (C) allocation within trees, thereby impairing tree growth. Recovery of root and leaf functioning and prioritized C supply to sink tissues after drought may compensate for drought-induced reduction of assimilation and growth. It remains unclear if C allocation to sink tissues during and following drought is controlled by altered sink metabolic activities or by the availability of new assimilates. Understanding such mechanisms is required to predict forests' resilience to a changing climate. We investigated the impact of drought and drought release on C allocation in a 100-y-old Scots pine forest. We applied 13 CO 2 pulse labeling to naturally dry control and long-term irrigated trees and tracked the fate of the label in above- and belowground C pools and fluxes. Allocation of new assimilates belowground was ca. 53% lower under nonirrigated conditions. A short rainfall event, which led to a temporary increase in the soil water content (SWC) in the topsoil, strongly increased the amounts of C transported belowground in the nonirrigated plots to values comparable to those in the irrigated plots. This switch in allocation patterns was congruent with a tipping point at around 15% SWC in the response of the respiratory activity of soil microbes. These results indicate that the metabolic sink activity in the rhizosphere and its modulation by soil moisture can drive C allocation within adult trees and ecosystems. Even a subtle increase in soil moisture can lead to a rapid recovery of belowground functions that in turn affects the direction of C transport in trees., Competing Interests: The authors declare no competing interest.

Long generation times have been suggested to hamper rapid genetic adaptation of organisms to changing environmental conditions. We examined if environmental memory of the parental Scots pines (Pinus sylvestris L.) drive offspring survival and growth. We used seeds from trees growing under naturally dry conditions (control), irrigated trees (irrigated from 2003 to 2016), and formerly irrigated trees ("irrigation stop"; irrigated from 2003-2013; control condition since 2014). We performed two experiments, one under controlled greenhouse conditions and one at the experimental field site. In the greenhouse, the offspring from control trees exposed regularly to drought were more tolerant to hot-drought conditions than the offspring from irrigated trees and showed lower mortality even though there was no genetic difference. However, under optimal conditions (high water supply and full sunlight), these offspring showed lower growth and were outperformed by the offspring of the irrigated trees. This different offspring growth, with the offspring of the "irrigation-stop" trees showing intermediate responses, points to the important role of transgenerational memory for the long-term acclimation of trees. Such memory effects, however, may be overridden by climatic extremes during germination and early growth stages such as the European 2018 mega-drought that impacted our field experiment., (© 2020 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

The 18 O signature of atmospheric water vapour (δ 18 O V ) is known to be transferred via leaf water to assimilates. It remains, however, unclear how the 18 O-signal transfer differs among plant species and growth forms. We performed a 9-hr greenhouse fog experiment (relative humidity ≥ 98%) with 18 O-depleted water vapour (-106.7‰) on 140 plant species of eight different growth forms during daytime. We quantified the 18 O-signal transfer by calculating the mean residence time of O in leaf water (MRT LW ) and sugars (MRT Sugars ) and related it to leaf traits and physiological drivers. MRT LW increased with leaf succulence and thickness, varying between 1.4 and 10.8 hr. MRT Sugars was shorter in C 3 and C 4 plants than in crassulacean acid metabolism (CAM) plants and highly variable among species and growth forms; MRT Sugars was shortest for grasses and aquatic plants, intermediate for broadleaf trees, shrubs, and herbs, and longest for conifers, epiphytes, and succulents. Sucrose was more sensitive to δ 18 O V variations than other assimilates. Our comprehensive study shows that plant species and growth forms vary strongly in their sensitivity to δ 18 O V variations, which is important for the interpretation of δ 18 O values in plant organic material and compounds and thus for the reconstruction of climatic conditions and plant functional responses., (© 2019 John Wiley & Sons Ltd.)

While photosynthetic isotope discrimination is well understood, the postphotosynthetic and transport-related fractionation mechanisms that influence phloem and subsequently tree ring δ 13 C are less investigated and may vary among species. We studied the seasonal and diel courses of leaf-to-phloem δ 13 C differences of water-soluble organic matter (WSOM) in vertical crown gradients and followed the assimilate transport via the branches to the trunk phloem at breast height in European beech (Fagus sylvatica) and Douglas fir (Pseudotsuga menziesii). δ 13 C of individual sugars and cyclitols from a subsample was determined by compound-specific isotope analysis. In beech, leaf-to-phloem δ 13 C differences in WSOM increased with height and were partly caused by biochemical isotope fractionation between leaf compounds. 13 C-Enrichment of phloem sugars relative to leaf sucrose implies an additional isotope fractionation mechanism related to leaf assimilate export. In Douglas fir, leaf-to-phloem δ 13 C differences were much smaller and isotopically invariant pinitol strongly influenced leaf and phloem WSOM. Trunk phloem WSOM at breast height reflected canopy-integrated δ 13 C in beech but not in Douglas fir. Our results demonstrate that leaf-to-phloem isotope fractionation and δ 13 C mixing patterns along vertical gradients can differ between tree species. These effects have to be considered for functional interpretations of trunk phloem and tree ring δ 13 C., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Elevated CO 2 along with rising temperature and water deficits can lead to changes in tree physiology and leaf biochemistry. These changes can increase heat- and drought-induced tree mortality. We aim to reveal the impacts of climatic drivers on individual compounds at the leaf level among European larch (Larix decidua) and mountain pine (Pinus mugo) trees, which are widely distributed at high elevations. We investigated seasonal carbon isotope composition (δ 13 C) and concentration patterns of carbohydrates and organic acids in needles of these two different species from a case study in the Swiss National Park (SNP). We found that average and minimum air temperatures were the main climatic drivers of seasonal variation of δ 13 C in sucrose and glucose as well as in concentrations of carbohydrates and citric acid/citrate in needles of both tree species. The impact of seasonal climatic drivers on larch and mountain pine trees at the needle level is in line with our earlier study in this region for long-term changes at the tree-ring level. We conclude that the species-specific changes in δ 13 C and concentrations of carbohydrates and organic acids are sensitive indicators of changes in the metabolic pathways occurring as a result of climatic changes., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Carbon isotope (13C) fractionations occurring during and after photosynthetic CO2 fixation shape the carbon isotope composition (δ13C) of plant material and respired CO2. However, responses of 13C fractionations to diel variation in starch metabolism in the leaf are not fully understood. Here we measured δ13C of organic matter (δ13COM), concentrations and δ13C of potential respiratory substrates, δ13C of dark-respired CO2 (δ13CR), and gas exchange in leaves of starch-deficient plastidial phosphoglucomutase (pgm) mutants and wild-type plants of four species (Arabidopsis thaliana, Mesembryanthemum crystallinum, Nicotiana sylvestris, and Pisum sativum). The strongest δ13C response to the pgm-induced starch deficiency was observed in N. sylvestris, with more negative δ13COM, δ13CR, and δ13C values for assimilates (i.e. sugars and starch) and organic acids (i.e. malate and citrate) in pgm mutants than in wild-type plants during a diel cycle. The genotype differences in δ13C values could be largely explained by differences in leaf gas exchange. In contrast, the PGM-knockout effect on post-photosynthetic 13C fractionations via the plastidic fructose-1,6-bisphosphate aldolase reaction or during respiration was small. Taken together, our results show that the δ13C variations in starch-deficient mutants are primarily explained by photosynthetic 13C fractionations and that the combination of knockout mutants and isotope analyses allows additional insights into plant metabolism., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Our understanding of how temporal variations of atmospheric water vapour and its isotopic composition (δ 18 O V ) influence water and assimilates in plants remains limited, restricting our ability to use δ 18 O as a tracer of ecophysiological processes. We exposed oak (Quercus robur) saplings under wet and dry soil moisture conditions to 18 O-depleted water vapour (c. - 200‰) at high relative humidity (c. 93%) for 5 h, simulating a fog event. We then traced the step change in δ 18 O V into water and assimilates (e.g. sucrose, hexoses, quercitol and starch) in the leaf lamina, main veins and twigs over 24 h. The immediate δ 18 O V effect was highest for δ 18 O of leaf lamina water, but 40% lower on δ 18 O of main vein water. To a smaller extent, we also observed changes in δ 18 O of twig xylem water. Depending on the individual assimilation rate of each plant, the 18 O-label was partitioned among different assimilates, with highest changes in δ 18 O of starch/sucrose and lowest in δ 18 O of quercitol. Additionally, 18 O-label partitioning and allocation towards leaf starch and twig phloem sugars was influenced by the plant water status. Our results have important implications for water isotope heterogeneity in plants and for our understanding of how the δ 18 O signal is incorporated into biomarkers., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Rationale: Levoglucosan is formed from cellulose during biomass burning. It is therefore often used as a specific tracer to quantify the contribution of wood burning to the aerosol loading. The stable oxygen isotope composition (δ 18 O value) of biomass is determined by the water cycle and varies regionally, and hence the δ 18 O value of levoglucosan could help to identify source regions of organic aerosols., Methods: After solvent extraction of the organic fraction and concentration steps, a recently developed methylation derivatisation technique was applied on experimental (i.e. controlled wood-burning experiments) and on ambient aerosol samples from Switzerland and Lithuania. The method achieves sufficient compound separation for isotope analysis in atmospheric particulate matter, enabling δ 18 O analysis of levoglucosan by gas chromatography/pyrolysis-isotope ratio mass spectrometry (GC/Pyr-IRMS), with a precision better than 1.0 ‰ and an accuracy of 0.3 ‰., Results: The δ 18 O value of the levoglucosan released during controlled wood-burning experiments was not significantly different from the cellulose δ 18 O values, which implies very little or no isotope fractionation during wood burning under the given conditions. While the δ 18 O values of levoglucosan in Swiss samples were as expected for the source region, those in Lithuania were 1-4 ‰ lower than expected. This may be due to differences in vegetation (grass vs wood) or burning conditions (high vs low temperatures)., Conclusions: Low oxygen isotope fractionation between cellulose and levoglucosan and clear differences in levoglucosan δ 18 O values between the Swiss and Lithuanian ambient samples demonstrate that our new method is useful for source appointment studies on wood-burning-derived aerosols., (Copyright © 2017 John Wiley & Sons, Ltd.)