Search

During prolonged dry periods, non-rainfall water (NRW) plays a vital role as water input into temperate grasslands, affecting the leaf surface water balance and plant water status. Previous chamber and laboratory experiments investigated air–leaf water exchange during dew deposition, but overlooked the importance of radiative cooling on air–leaf water exchange because the chamber is a heat trap, preventing radiative cooling. To complement these previous studies, we conducted a field study, in which we investigated the effect of radiatively-induced NRW inputs on leaf water isotope signals and air–leaf water exchange in a temperate grassland during the dry-hot summers of 2018 and 2019. We carried out field measurements of the isotope composition of atmospheric water vapor, NRW droplets on foliage, leaf water, xylem water of root crown, and soil water, combined with meteorological and plant physiological measurements. We combined radiation measurements with thermal imaging to estimate leaf temperatures using different methods, and computed the corresponding leaf conductance and air–leaf water exchange. Our results indicate that radiative cooling and leaf wetting induced a switch of direction in the net water vapor exchange from leaf-to-air to air-to-leaf. The leaf conductance and air–leaf water exchange varied by species due to the species-specific biophysical controls. Our results highlight the ecological relevance of radiative cooling and leaf wetting in natural temperate grasslands, a process which is expected to influence land surface water budgets and may impact plant survival in many regions in a drier climate., Agricultural and Forest Meteorology, 328, ISSN:0168-1923, ISSN:1873-2240

Above and belowground compartments in ecosystems are closely coupled on daily to annual timescales. In mature forests, this interlinkage and how it is impacted by drought is still poorly understood. Here, we pulse‐labelled 100‐year‐old trees with 13CO2 within a 15‐year‐long irrigation experiment in a naturally dry pine forest to quantify how drought regime affects the transfer and use of assimilates from trees to the rhizosphere and associated microbial communities. It took 4 days until new 13C‐labelled assimilates were allocated to the rhizosphere. One year later, the 13C signal of the 3‐h long pulse labelling was still detectable in stem and soil respiration, which provides evidence that parts of the assimilates are stored in trees before they are used for metabolic processes in the rhizosphere. Irrigation removing the natural water stress reduced the mean C residence time from canopy uptake until soil respiration from 89 to 40 days. Moreover, irrigation increased the amount of assimilates transferred to and respired in the soil within the first 10 days by 370%. A small precipitation event rewetting surface soils altered this pattern rapidly and reduced the effect size to +35%. Microbial biomass incorporated 46 ± 5% and 31 ± 7% of the C used in the rhizosphere in the dry control and irrigation treatment respectively. Mapping the spatial distribution of soil‐respired 13CO2 around the 10 pulse‐labelled trees showed that tree rhizospheres extended laterally 2.8 times beyond tree canopies, implying that there is a strong overlap of the rhizosphere among adjacent trees. Irrigation increased the rhizosphere area by 60%, which gives evidence of a long‐term acclimation of trees and their rhizosphere to the drought regime. The moisture‐sensitive transfer and use of C in the rhizosphere has consequences for C allocation within trees, soil microbial communities and soil carbon storage., To quantify the transfer of carbon from tree canopies into the belowground, we conducted a pulse‐labelling with 100‐year old trees in a 15‐year‐long irrigation experiment within a naturally dry pine forest. It took four days until new assimilates were allocated from tree canopies to the rhizosphere. One year later, the 13C signal of the three‐hour long pulse labelling was still detectable in stem and soil respiration. Mapping the spatial distribution of soil‐respired 13CO2 around pulse‐labelled trees showed that rhizospheres extended laterally 2.8 times beyond tree canopies. Drought reduced the transfer rates and spatial distribution of assimilates allocated to the soil.

Plants have evolved to grow under prominently fluctuating environmental conditions. In experiments under controlled conditions, temperature is often set to artificial, binary regimes with constant values at day and at night. This study investigated how such a diel (24 hr) temperature regime affects leaf growth, carbohydrate metabolism and gene expression, compared to a temperature regime with a field‐like gradual increase and decline throughout 24 hr. Soybean (Glycine max) was grown under two contrasting diel temperature treatments. Leaf growth was measured in high temporal resolution. Periodical measurements were performed of carbohydrate concentrations, carbon isotopes as well as the transcriptome by RNA sequencing. Leaf growth activity peaked at different times under the two treatments, which cannot be explained intuitively. Under field‐like temperature conditions, leaf growth followed temperature and peaked in the afternoon, whereas in the binary temperature regime, growth increased at night and decreased during daytime. Differential gene expression data suggest that a synchronization of cell division activity seems to be evoked in the binary temperature regime. Overall, the results show that the coordination of a wide range of metabolic processes is markedly affected by the diel variation of temperature, which emphasizes the importance of realistic environmental settings in controlled condition experiments., Dynamically changing temperature affects soybean leaf growth and regulatory mechanisms in non‐intuitive ways. The results may help improving knowledge transfer between experiments performed under controlled conditions and experiments performed in the field.

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 (CO2)-C-13 pulse labeling to naturally dry control and long-term irrigated trees and tracked the fate of the label in aboveand 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.

The carbon isotopic composition (δ13C) of foliage is often used as proxy for plant performance. However, the effect of NO3– vs. NH4+ supply on δ13C of leaf metabolites and respired CO2 is largely u...

N2O is a stratospheric ozone depleting substance and a potent greenhouse gas which significantly contributes to global warming. Although soils are the largest source of N2O emissions, knowledge gaps in the understanding of N2O production and reduction processes in soils still exist. Here, we investigated N2O production and consumption processes along soil depth profiles in a mesocosm experiment using natural-abundance N2O and NO3- isotopic signatures as well as abundances of soil microbial genes associated with N2O production (nirK, nirS) and reduction (nosZ). Soil columns either displayed undisturbed soil stratification (control treatments), or contained an artificial clay layer at 35 cm depth (clay treatment), which acted as a diffusion barrier and thus induced O2-limited conditions in deeper strata. We collected soil pore gas, soil solution and soil samples at five depths of the soil columns over the course of four weeks. In addition, we continuously monitored N2O fluxes at the soil surface and soil environmental parameters (oxygen, moisture, temperature) along the soil depth profiles. Microbial gene analysis in soil samples revealed similar abundances of nirK, nirS and nosZ in the two treatments across the entire soil depth profiles. The distribution of the functional genes was thus not indicative of enhanced N2O production and/or reduction in O2-limited conditions. However, lowest O2 concentrations below the clay layer were associated with highest 15N and 18O enrichments in both NO3- and N2O, indicating N2O production by denitrification and fractional N2O reduction. In addition, we found higher N2O concentrations and surface fluxes for the clay treatment. Our observations imply a dominance of N2O production over N2O reduction, even under conditions most favorable for complete denitrification.

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.

The intention of this chapter is to give insight into the properties and peculiarities of the stable isotopes of the bioelements. Following an overview about the terminology and ʻtechnical jargonʼ used in stable isotope sciences, methods to calculate and express isotopic abundances are presented. Subsequently, a short description of the physicochemical basis of equilibrium and kinetic (mass-dependent) isotope effects (EIEs and KIEs) as origin of isotope fractionation in chemical and biological systems is given. Further, measures for calculation and presentation of isotope fractionation are introduced and the corresponding properties of these quantities are critically discussed. Finally, examples for equilibrium and kinetic isotope fractionation in biochemical reactions are presented in more details and subsequent effects and consequences including the relationship between EIEs and KIEs are reviewed.

Quality assuranceand quality control(QA/QC) are important components of every study. In this chapter, we give an overview of QA/QC specific for tree-ring stable-isotope analysis from the perspective of the entire research project, rather than from the operation of Isotope Ratio Mass Spectrometers (IRMS). We address how users of stable isotope tree-ring data can quantify the quality of their data for reporting in publications by calculating accuracy and precision. We cover some of the potential sources of error that can occur during sample processing and isotopic measurements, basic principles of calibration to the appropriate isotopic scales, and how researchers can detect error and calculate uncertaintyusing duplicatesand quality control standards.

Nitrous oxide (N

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

Purpose: Mediterranean wood pastures are the largest agroforestry system in Europe. Traditional silvo-pastoral uses shaped these systems into a mosaic of trees and open grassland. However, little is known about how this structure may influence ecosystem carbon (C) and nitrogen (N) dynamics, considering different canopy types and interactions with the herbaceous layer.Aims: To unravel the canopy effect on C and N dynamics (1) under representative canopy types, including traditional Quercus stands and Pinus pinea L. plantations; and (2) in interaction with the herbaceous plant functional types (PFT).Methods: We analysed C and N content and the isotopic natural abundance on soil and plant materials at the open grassland and under the different tree canopies.Results: Under the canopy, soil C and N content, and N availability for the herbaceous layer increased. The canopy effect performed differences between Quercus and P. pinea plots, as detected on the belowground biomass C content. Also, the canopy effect was more pronounced with increasing environmental constraints, highlighting the relevance of trees in more restrictive environments. PFT ecophysiological characteristics interacted with tree canopies, as reflected the enhanced efficiency taking-up N, and/or exploiting symbiotic N of grasses, which was probably one of the causes of their dominancy under the canopy, at higher N availability.Conclusion: Changes in the tree coverage and tree species will significantly change ecosystem C and N storage and cycling. Therefore, it is highly advisable to preserve a balance between trees and open grassland, and traditional Quercus stands, to keep ecosystem services provisioning.

Habitats with fluctuating resource conditions pose specific challenges to plants, and they often favor a small subset of species that includes exotic invaders. These species must possess a superior ability to capitalize on resource pulses through faster resource uptake or greater resource-use efficiency. We addressed this question in an experiment with invasive knotweed, a noxious invader of temperate ecosystems that is known to benefit from nutrient fluctuations. We used stable isotopes to track the uptake and use efficiency of a nitrogen pulse in competition pairs between knotweed and five native competitors. We found that nitrogen pulses indeed promoted knotweed invasion and that this is explained by a superior efficiency in turning the taken-up extra nitrogen into biomass, rather than capturing an overproportional share of the nitrogen. Thus, temporary increases in nutrient availability might help knotweed to invade natural environments, such as river banks or nitrogen-polluted margins and wastelands, where nutrient fluctuations occur. Our experiment shows that resource-use efficiency can drive invasion under fluctuating resource conditions, and that stable isotopes help to understand these processes.

Stable isotopes at natural abundance are key tools to study physiological processes occurring outside the temporal scope of manipulation and monitoring experiments. Whole-molecule carbon isotope ratios (13C/12C) enable assessments of plant carbon uptake yet conceal information about carbon allocation. Here, we identify an intramolecular 13C/12C signal at tree-ring glucose C-5 and C-6 and develop experimentally testable theories on its origin. More specifically, we assess the potential of processes within C3 metabolism for signal introduction based (inter alia) on constraints on signal propagation posed by metabolic networks. We propose that the intramolecular signal reports carbon allocation into major metabolic pathways in actively photosynthesizing leaf cells including the anaplerotic, shikimate, and non-mevalonate pathway. We support our theoretical framework by linking it to previously reported whole-molecule 13C/12C increases in cellulose of ozone-treated Betula pendula and a highly significant relationship between the intramolecular signal and tropospheric ozone concentration. Our theory postulates a pronounced preference for leaf cytosolic triose-phosphate isomerase to catalyse the forward reaction in vivo (dihydroxyacetone phosphate to glyceraldehyde 3-phosphate). In conclusion, intramolecular 13C/12C analysis resolves information about carbon uptake and allocation enabling more comprehensive assessments of carbon metabolism than whole-molecule 13C/12C analysis., Journal of Experimental Botany, 73 (8), ISSN:1460-2431, ISSN:0022-0957

Studies using carbon isotope differences between C

Within the plant and earth sciences, stable isotope analysis is a versatile tool conveying information (inter alia) about plant physiological and paleoclimate variability across scales. Here, we identify a 13C signal (i.e., systematic 13C/ 12C variation) at tree-ring glucose C-4 and report an experimentally testable theory on its origin. We propose the signal is introduced by glyceraldehyde-3-phosphate dehydrogenases in the cytosol of leaves. It conveys two kinds of (potentially convoluted) information: (i) commitment of glyceraldehyde 3-phosphate to 3-phosphoglycerate vs. fructose 1,6-bisphosphate metabolism, and (ii) the contribution of non-phosphorylating vs. phosphorylating glyceraldehyde-3-phosphate dehydrogenase to catalysing the glyceraldehyde 3-phosphate to 3-phosphoglycerate forward reaction of glycolysis. Theory is supported by 13C fractionation modelling. Modelling results provide first evidence in support of the cytosolic oxidation-reduction (COR) cycle, a carbon-neutral mechanism supplying NADPH at the expense of ATP and NADH which may help to maintain leaf-cytosolic redox balances. In line with expectations related to COR cycling, we found a positive correlation between air vapour pressure deficit and 13C discrimination at glucose C-4. Overall, 13C-4 signal analysis may enable an improved understanding of leaf carbon and energy metabolism., Journal of Experimental Botany, 72 (20), ISSN:1460-2431, ISSN:0022-0957

Rationale The oxygen isotopic composition (here shown as the δ 18O value) of soluble sugars in leaves and phloem tissue holds valuable information about plant functions in response to climatic changes. However, δ 18O 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 18O 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 18O 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 18O values; yet the purified leaf and phloem sugars possessed higher δ 18O values than the fraction of water‐soluble compounds. Moreover, the exudation time significantly modulated the δ 18O 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 18O values of phloem sugars. Conclusions We show that the sample preparation of plant sugars for the reliable determination of δ 18O 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., Rapid Communications in Mass Spectrometry, 34 (19), ISSN:1097-0231, ISSN:0951-4198

The carbon isotopic composition (δ

Soil respiration is an important carbon flux and key process determining the net ecosystem production of terrestrial ecosystems. To address the enormous lack of quantification and understanding of seasonality in soil respiration of tropical forests in the Congo Basin, soil CO2 fluxes and potential controlling factors were measured for the first time annually in two dominant forest types (lowland and montane) of the Congo Basin during three years at varying temporal resolution. Soil CO2 fluxes from the Congo Basin resulted in 3.69 ± 1.22 and 3.82 ± 1.15 µmol CO2 m−2 s−1 for lowland and montane forests, respectively. Respiration in montane forest soils showed a clear seasonality with decreasing flux rates during the dry season. Montane forest soil CO2 fluxes were positively correlated with soil moisture while CO2 fluxes in the lowland forest were not. Paired ẟ13C values of soil organic carbon (SOC) and soil CO2 indicated that SOC in lowland forests is more decomposed than in montane forests, suggesting that respiration is controlled by C availability rather than environmental factors. In general, C in montane forests was more enriched in 13C throughout the whole cascade of carbon intake via photosynthesis, litterfall, SOC, and soil CO2 compared to lowland forests, pointing to a more open system. Even though soil CO2 fluxes are similarly high in lowland and montane forests of the Congo Basin, the drivers of them were different, i.e. soil moisture for montane forest and C availability for lowland forest.

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.

Oxygen isotope analysis of plant material, such as sugars in different tissues, provides an important tool to understand how plants function, interact with their environment and also cope with climate change. Knowing how to extract and purify carbohydrates without artificially altering their oxygen isotope ratio (δ18O) is therefore essential.We aimed to resolve the impact of different steps on sugars' δ18O values during their extraction and purification from leaf and phloem tissue. More precisely, we investigated (1) different drying processes (oven- vs freeze-drying), and (2) how extraction and purification affect leaf sugars. To clearly see fractionation and exchange processes, these experiments were performed using 18O-labelled water. We further examined (3) the influence of different EDTA media and immersion times to facilitate sugar exudation and subsequent yield from twig phloem tissue. Finally, we analysed (4) the sugar phloem composition, as well as the individual compounds’ carbon isotopic signatures (δ13C).Comparison of freeze- and oven-dried sugars showed lower δ18O memory effects and more consistent oxygen isotopic signatures across different sugars, indicating lyophilisation as the more reliable method. The extraction and purification can be conducted without significant oxygen isotope fractionation. However, 18O-depletion was observed when sugars were dissolved and dried multiple times. This suggests that additional dissolution and drying steps should best be avoided whenever possible. Different immersion times and exudation media during twig phloem extraction revealed to have a substantial influence on the phloem sugars' overall oxygen isotopic signature, their composition, and the individual compounds' δ13C values.Our research illustrates which precautions during sample preparation – from drying to extracting and purifying – need to be taken when plant sugars and their oxygen isotopic signature are of interest. Regarding the preservation of the phloem sugars' original δ18O values and stabilising their composition (prevention of sucrose degradation) as much as possible, we recommend a short immersion time of approx. 1 hour. After a thorough initial rinse of the tissue, the sap should be eluted in pure water without any additives (no EDTA). This further reduces the possibility of hexoses to exchange oxygen with that of the surrounding water.

Over the past decades, average global wheat yields have increased by about 250 %, mainly due to the cultivation of high-yielding wheat cultivars. This selection process not only affected aboveground parts of plants, but in some cases also reduced the root biomass, with potentially large consequences for the amount of organic carbon (OC) transferred to the soil. To study the effect of wheat breeding for high-yielding cultivars on subsoil OC dynamics, two old and two new wheat cultivars from the Swiss wheat breeding program were grown for one growing season in 1.5 m-deep lysimeters and pulse-labelled with 13CO2, to quantify the amount of assimilated carbon that was transferred belowground and potentially stabilized in the soil. The results show that although the old wheat cultivars with higher root biomass transferred more assimilated carbon belowground compared to more recent cultivars, no significant differences in net soil organic carbon (SOC) stabilization were found between the different cultivars. As a consequence, the long-term effect of wheat cultivar selection on SOC stocks will depend on the amount of root biomass that is stabilized in the soil. Our results suggest that the process of wheat selection for high-yielding cultivars resulted in lower amounts of belowground carbon translocation, with potentially important effects on SOC stocks. Further research is necessary to quantify the long-term importance of this effect.

RATIONALE In the last few years, the study of N2 O site-specific nitrogen isotope composition has been established as a powerful technique to disentangle N2 O emission pathways. This trend has been accelerated by significant analytical progress in the field of isotope ratio mass spectrometry (IRMS) and more recently quantum cascade laser absorption spectroscopy (QCLAS). METHODS The ammonium nitrate (NH4 NO3 ) decomposition technique provides a strategy to scale the 15 N site-specific (SP ≡ δ15 Nα - δ15 Nβ ) and bulk (δ15 Nbulk = (δ15 Nα + δ15 Nβ )/2) isotopic composition of N2 O against the international standard for the 15 N/14 N isotope ratio (AIR-N2 ). Within the current project 15 N fractionation effects during thermal decomposition of NH4 NO3 on the N2 O site preference were studied using static and dynamic decomposition techniques. RESULTS The validity of the NH4 NO3 decomposition technique to link NH4+ and NO3- moiety-specific δ15 N analysis by IRMS to the site-specific nitrogen isotopic composition of N2 O was confirmed. However, the accuracy of this approach for the calibration of δ15 Nα and δ15 Nβ values was found to be limited by non-quantitative NH4 NO3 decomposition in combination with substantially different isotope enrichment factors for the conversion of the NO3- or NH4+ nitrogen atom into the α or β position of the N2 O molecule. CONCLUSIONS The study reveals that the completeness and reproducibility of the NH4 NO3 decomposition reaction currently confine the anchoring of N2 O site-specific isotopic composition to the international isotope ratio scale AIR-N2 . The authors suggest establishing a set of N2 O isotope reference materials with appropriate site-specific isotopic composition, as community standards, to improve inter-laboratory compatibility. Copyright © 2016 John Wiley & Sons, Ltd.

Leaf respiration in the dark and its C isotopic composition (δ(13) CR ) contain information about internal metabolic processes and respiratory substrates. δ(13) CR is known to be less negative compared to potential respiratory substrates, in particular shortly after darkening during light enhanced dark respiration (LEDR). This phenomenon might be driven by respiration of accumulated (13) C-enriched organic acids, however, studies simultaneously measuring δ(13) CR during LEDR and potential respiratory substrates are rare. We determined δ(13) CR and respiration rates (R) during LEDR, as well as δ(13) C and concentrations of potential respiratory substrates using compound-specific isotope analyses. The measurements were conducted throughout the diel cycle in several plant species under different environmental conditions. δ(13) CR and R patterns during LEDR were strongly species-specific and showed an initial peak, which was followed by a progressive decrease in both values. The species-specific differences in δ(13) CR and R during LEDR may be partially explained by the isotopic composition of organic acids (e.g., oxalate, isocitrate, quinate, shikimate, malate), which were (13) C-enriched compared to other respiratory substrates (e.g., sugars and amino acids). However, the diel variations in both δ(13) C and concentrations of the organic acids were generally low. Thus, additional factors such as the heterogeneous isotope distribution in organic acids and the relative contribution of the organic acids to respiration are required to explain the strong (13) C enrichment in leaf dark-respired CO2 .

Hydrogen (H) isotopes of plant organic compounds are rarely employed in ecological studies. If so, these values are interpreted as being indicative of the plant source and/or leaf water. Recent observations suggest, however, that variations in hydrogen isotope fractionation that occur during the biosynthesis of plant compounds (2H-ebio) imprint valuable metabolic information into the hydrogen isotope composition (δ2H values) of plant organic compounds. Here we show a consistent 2H-enrichment of compounds in heterotrophically growing plants across a series of autotrophic/heterotrophic plant pairs. We suggest that this is due to a higher recycling of compounds in the Calvin and tricarboxylic acid cycles in heterotrophic plants that is associated with a more complete exchange of C-bound H with the surrounding 2H-enriched foliar water. Interestingly, we found that 2H-enrichment in heterotrophic plants was larger for carbohydrates than for lipids, with an average 2H-enrichment of 76 ± 9‰ in α-cellulose and 23 ± 23‰ in n-alkanes. We propose that this systematically larger 2H-enrichment for carbohydrates than for lipids is either due to different level of 2H-fractionation associated with heterotrophically produced NADPH, or to the potential uptake of lipids by heterotrophic plants. With the work we present here, we contribute to a better mechanistic understanding of what the biochemical principles are that couple the carbohydrate dynamics of plants to their δ2H values and hope to foster as such the application of H isotopes in plant sciences.

Rapid Communications in Mass Spectrometry, 33 (13), ISSN:1097-0231, ISSN:0951-4198

Oversaturation of the Faraday cup amplifiers of isotope ratio mass spectrometers when using tracers that are highly enriched in heavier isotopes (up to 99.9%) remains a major bottleneck to obtaining high-precision measurements. The memory effect plays a key role in reducing tracer sample measurement precision and accuracy. Several sample preparation approaches are known to reduce memory effects and to improve tracer sample measurement precision. However, the potential benefits when using very high enrichment tracer samples (+1000 mUr) have not been tested.In this study, we test how specific sample positioning for measurements and frequent use of natural isotope abundance reference materials within the sequence affects the precision and accuracy of isotopic ratio analyses when using a Flash elemental analyser coupled to a DeltaFrequent use of natural isotope abundance materials and specific positioning increasedWe conclude that highly enriched tracer samples do not require dilution before analysis. Tracer sample precision can be improved by using a specific measurement order of expected isotope abundance and by the frequent use of natural abundance reference materials.

Habitats with fluctuating resource conditions pose specific challenges to plants, and they often favor a small subset of species that includes exotic invaders. These species must possess a superior ability to capitalize on resource pulses through faster resource uptake or greater resource-use efficiency. We addressed this question in an experiment with invasive knotweed, a noxious invader of temperate ecosystems that is known to benefit from nutrient fluctuations. We used stable isotopes to track the uptake and use efficiency of a nitrogen pulse in competition pairs between knotweed and five native competitors. We found that nitrogen pulses indeed promoted knotweed invasion and that this is explained by a superior efficiency in turning the taken-up extra nitrogen into biomass, rather than capturing an overproportional share of the nitrogen. Thus, temporary increases in nutrient availability might help knotweed to invade natural environments, such as river banks or nitrogen-polluted margins and wastelands, where nutrient fluctuations occur. Our experiment shows that resource-use efficiency can drive invasion under fluctuating resource conditions, and that stable isotopes help to understand these processes.

Rationale Oxygen isotope fractionation of molecular O2 is an important process for the study of aerobic metabolism, photosynthesis, and formation of reactive oxygen species. The latter is of particular interest for investigating the mechanism of enzyme-catalyzed reactions, such as the oxygenation of organic pollutants, which is an important detoxification mechanism. Methods We developed a simple method to measure the δ18O values of dissolved O2 in small samples using automated split injection for gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). After creating a N2 headspace, the dissolved O2 partitions from aqueous solution to the headspace, from which it can be injected into the gas chromatograph. Results In aqueous samples of 10 mL and in diluted air samples, we quantified the δ18O values at O2 concentrations of 16 μM and 86 μM, respectively. The chromatographic separation of O2 and N2 with a molecular sieve column made it possible to use N2 as the headspace gas for the extraction of dissolved O2 from water. We were therefore able to apply a rigorous δ18O blank correction for the quantification of 18O/16O ratios in 20 nmol of injected O2. Conclusions The successful quantification of 18O-kinetic isotope effects associated with enzymatic and chemical reduction of dissolved O2 illustrates how the proposed method can be applied for studying enzymatic O2 activation mechanisms in a variety of (bio)chemical processes. Copyright © 2016 John Wiley & Sons, Ltd.

Summary: Hydrogen (H) isotope ratio (δ2H) analyses of plant organic compounds have been applied to assess ecohydrological processes in the environment despite a large part of the δ2H variability observed in plant compounds not being fully elucidated. We present a conceptual biochemical model based on empirical H isotope data that we generated in two complementary experiments that clarifies a large part of the unexplained variability in the δ2H values of plant organic compounds. The experiments demonstrate that information recorded in the δ2H values of plant organic compounds goes beyond hydrological signals and can also contain important information on the carbon and energy metabolism of plants. Our model explains where 2H‐fractionations occur in the biosynthesis of plant organic compounds and how these 2H‐fractionations are tightly coupled to a plant's carbon and energy metabolism. Our model also provides a mechanistic basis to introduce H isotopes in plant organic compounds as a new metabolic proxy for the carbon and energy metabolism of plants and ecosystems. Such a new metabolic proxy has the potential to be applied in a broad range of disciplines, including plant and ecosystem physiology, biogeochemistry and palaeoecology.

During two extended summer seasons in 2006 and 2007 we operated two battery driven versions of the Caltech active strand cloud water collector (MiniCASCC) at the Niesen mountain (2362 m a.s.l.) in the northern part of the Swiss Alps, and two devices at the Lägeren research tower (690 m a.s.l.) at the northern boundary of the Swiss Plateau. During these two field operation phases we gained weekly samples of fog water, where we analyzed the major anions and cations, and the isotope ratios of fog water (in form of δ2H and δ18O). Dominant ions in fog water at all sites were NH4+, NO3−, and SO42 −. Compared to precipitation, the enrichment factors in fog water were in the range 5–9 at the highest site, Niesen Kulm. We found considerably lower summertime ion loadings in fog water at the two Alpine sites than at lower elevations above the Swiss Plateau. The lowest ion concentrations were found at the Niesen Kulm site at 2300 m a.s.l., whereas the highest concentrations (a factor 7 compared to Niesen Kulm) were found in fog water at the Lägeren site. Occult nitrogen deposition was estimated from fog frequency and typical fog water flux rates. This pathway contributes 0.3–3.9 kg N ha− 1 yr− 1 to the total N deposition at the highest site on Niesen mountain, and 0.1–2.2 kg N ha− 1 yr− 1 at the lower site. These inputs are the reverse of ion concentrations measured in fog due to the 2.5 times higher frequency of fog occurrence at the mountain top (overall fog occurrence was 25% of the time) as compared to the lower Niesen Schwandegg site. Although fog water concentrations were on the lower range reported in earlier studies, fog water is likely to be an important N source for Northern Alpine ecosystems and might reach values up to 16% of the total N deposition and up to 75% of wet N deposition by precipitation.

Carbon isotope composition in respired CO2 and organic matter of individual organs were measured on peanut seedlings during early ontogeny in order to compare fractionation during heterotrophic growth and transition to autotrophy in a species with lipid seed reserves with earlier results obtained on beans. Despite a high lipid content in peanut seeds (48 %) compared with bean seeds (1.5 %), the isotope composition of leaf- and root-respired CO2 as well as its changes during ontogeny were similar to already published data on bean seedlings: leaf-respired CO2 became C-13-enriched reaching -21.5 parts per thousand, while root-respired CO2 became C-13-depleted reaching around -31 parts per thousand at the four-leaf stage. The opposite respiratory fractionation in leaves vs. roots already reported for C-3 herbs was thus confirmed for peanuts. However, contrarily to beans, the peanut cotyledon-respired CO2 was markedly C-13-enriched, and its C-13-depletion was noted from the two-leaf stage onwards only. Carbohydrate amounts being very low in peanut seeds, this cannot be attributed solely to their use as respiratory substrate. The potential role of isotope fractionation during glyoxylate cycle and/or gluconeogenesis on the C-13-enriched cotyledon-respired CO2 is discussed.

We developed a small version of the Caltech active strand cloud water collector (CASCC) for biogeochemical investigations in ecological applications. The device is battery powered and thus allows operation at locations where mains power is not available. The collector is designed for sampling periods of up to one week, depending on fog frequency. Our new device is equipped with standard sensors for air temperature, relative humidity, wind, and horizontal visibility for fog detection with a low-cost optical sensor. In mountain areas and during times when clouds are thin the installation of the visibility sensor became a key issue, which limits the potential to estimate liquid water content of the sampled fog. Field tests with 5 devices at three different sites in the Swiss Alps (Niesen) and the Jura Mountains (Lageren, Switzerland) during two extended summer seasons in 2006 and 2007 showed that in almost all cases it was possible to obtain sample volumes which were large enough for the examination of basic inorganic chemistry of the collected cloud water. Collection rates varied typically from 12 to 30 mL h− 1. The fog droplet cutoff diameter is ≈ 6 μm, which is low enough to include all droplet sizes that are relevant for the liquid water content of typical fog types in the collected samples. From theoretical assumptions of the collection efficiency and theoretical droplet spectra it is possible to estimate the liquid water content of the sampled fog or cloud. Our new fog collector can be constructed and operated at relatively low costs. In combination with chemical and isotopic analyses of the sampled water, this allows to quantify nutrient and pollutant fluxes as is typically needed in ecosystem biogeochemistry studies.

Although the instrumental coupling of gas chromatography pyrolysis isotope ratio mass spectrometry (GC Py IRMS) for compound specific O 18 analysis has been commercially available for more than a decade this method has been hardly applied so far. Here we present the first GC Py IRMS O 18 results for trimethylsilyl derivatives of plant sap relevant sugars and a polyalcohol (glucose fructose sucrose raffinose and pinitol). Particularly we focus on sucrose which is assimilated in leaves and which is the most important transport sugar in plants and hence of utmost relevance in plant physiology and paleoclimate studies. Replication measurements of sucrose standards and concentration series indicate that the GC Py IRMS O 18 measurements are not stable over time and that they are amount (area) dependent. We therefore suggest running sample batch replication measurements in alternation with standard concentration series of reference material. This allows for carrying out (i) a drift correction (ii) a calibration against reference material and (iii) an amount (area) correction. Tests with O 18 enriched water do not provide any evidence for oxygen isotope exchange reactions affecting sucrose and raffinose. We present the first application of GC Py IRMS O 18 analysis for sucrose from needle extract (soluble carbohydrate) samples. The obtained O 18(sucrose/ Vienna Standard Mean Ocean Water (VSMOW)) values are more positive and vary in a wider range (32.1 40.1 parts per thousand) than the O 18(bulk/ VSMOW) values (24.6 27.2 parts per thousand). Furthermore they are shown to depend on the climate parameters maximum day temperature relative air humidity and cloud cover. These findings suggest that O 18(sucrose) of the investigated needles very sensitively reflects the climatically controlled evaporative O 18 enrichment of leaf water and thus highlights the great potential of GC Py IRMS O 18(sucrose) analysis for plant physiology and paleoclimate studies.

An international project developed, quality-tested, and determined isotope-delta values of 19 new organic reference materials (RMs) for hydrogen, carbon, and nitrogen stable isotope-ratio measurements, in addition to analyzing pre-existing RMs NBS 22 (oil), IAEA-CH-7 (polyethylene foil), and IAEA-600 (caffeine). These new RMs enable users to normalize measurements of samples to isotope-delta scales. The RMs span a range of delta H-2(VSMOW-SLAP) values from -210.8 to +397.0 mUr or parts per thousand, for delta C-13(VPDB-LSVEC) from -40.81 to +0.49 mUr and for delta N-15(Air) from -5.21 to +61.53 mUr. Many of the new RMs are amenable to gas and liquid chromatography. The RMs include triads of isotopically contrasting caffeines, C-16 n-alkanes, n-C-20-fatty acid methyl esters (FAMEs), glycines, and L-valines, together with polyethylene powder and string, one n-C-17-FAME, a vacuum oil (NBS 22a) to replace NBS 22 oil, and a H-2-enriched vacuum oil. A total of 11 laboratories from 7 countries used multiple analytical approaches and instrumentation for 2-point isotopic normalization against international primary measurement standards. The use of reference waters in silver tubes allowed direct normalization of delta H-2 values of organic materials against isotopic reference waters following the principle of identical treatment. Bayesian statistical analysis yielded the mean values reported here. New RMs are numbered from USGS61 through USGS78, in addition to NBS 22a. Because of exchangeable hydrogen, amino acid RMs currently are recommended only for carbon- and nitrogen-isotope measurements. Some amino acids contain C-13 and carbon-bound organic H-2-enrichments at different molecular sites to provide RMs for potential site-specific isotopic analysis in future studies.

Summary How will carbon source–sink relations of 35-yr-old larch trees (Larix decidua) at the alpine treeline respond to changes in atmospheric CO2 and climate? We evaluated the effects of previously elevated CO2 concentrations (9 yr, 580 ppm, ended the previous season) and ongoing soil warming (4 yr, + 4°C). Larch branches were pulse labeled (50 at% 13CO2) in July 2010 to trace fresh assimilates through tissues (buds, needles, bark and wood) and non-structural carbon compounds (NCC; starch, lipids, individual sugars) using compound-specific isotope analysis. Nine years of elevated CO2 did not lead to increased NCC concentrations, nor did soil warming increase NCC transfer velocities. By contrast, we found slower transfer velocities and higher NCC concentrations than reported in the literature for lowland larch. As a result of low dilution with older carbon, sucrose and glucose showed the highest maximum 13C labels, whereas labels were lower for starch, lipids and pinitol. Label residence times in needles were shorter for sucrose and starch (c. 2 d) than for glucose (c. 6 d). Although our treatments showed no persistent effect on larch carbon relations, low temperature at high altitudes clearly induced a limitation of sink activities (growth, respiration, root exudation), expressed in slower carbon transfer and higher NCC concentrations.

In this work, we present the first observations of stable water isotopologue ratios in cloud droplets of different sizes collected simultaneously. We address the question whether the isotope ratio of droplets in a liquid cloud varies as a function of droplet size. Samples were collected from a ground intercepted cloud (= fog) during the Hill Cap Cloud Thuringia 2010 campaign (HCCT-2010) using a three-stage Caltech Active Strand Cloud water Collector (CASCC). An instrument test revealed that no artificial isotopic fractionation occurs during sample collection with the CASCC. Furthermore, we could experimentally confirm the hypothesis that the δ values of cloud droplets of the relevant droplet sizes (μm-range) were not significantly different and thus can be assumed to be in isotopic equilibrium immediately with the surrounding water vapor. However, during the dissolution period of the cloud, when the supersaturation inside the cloud decreased and the cloud began to clear, differences in isotope ratios of the different droplet sizes tended to be larger. This is likely to result from the cloud's heterogeneity, implying that larger and smaller cloud droplets have been collected at different moments in time, delivering isotope ratios from different collection times., Atmospheric Chemistry and Physics, 12 (20), ISSN:1680-7375, ISSN:1680-7367

The δ(18)O value of the p-hydroxy group of L-tyrosine depends on the biosynthesis by plants or animals, respectively. In animal proteins it reflects the diet and is therefore an absolute indicator for illegal feeding with meat and bone meal. The aim of this investigation was to perform the positional (18)O determination on L-tyrosine via a one-step enzymatic degradation. Proteins from plants, herbivores, omnivores, and carnivores were characterized by their δ(13)C, δ(15)N, and δ(18)O values, the latter for normalizing the positional δ(18)O values. Their L-tyrosine was degraded by tyrosine phenol lyase to phenol, analyzed as (2,4,6)-tribromophenol. Degradation by tyrosine decarboxylase yielded tyramine. The δ(18)O values of both analytes corresponded to the trophic levels of their sources but were not identical, probably due to an isotope effect on the tyrosine phenol lyase reaction. Availability of the enzyme, easy control of the reaction, and isolation of the analyte are in favor of tyrosine decarboxylase degradation as a routine method.

Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures reflect external versus internal limitations of CO2 fixation. There has been recent renewed interest in the isotope composition of carbohydrates, mainly for use in CO2 flux partitioning studies at the ecosystem level. The major obstacle to the use of carbohydrates in such studies has been the lack of an acknowledged method to isolate starch and soluble sugars for isotopic measurements. We here report on the comparison and evaluation of existing methods (acid and enzymatic hydrolysis for starch; ion-exchange purification and compound-specific analysis for sugars). The selectivity and reproducibility of the methods were tested using three approaches: (i) an artificial leaf composed of a mixture of isotopically defined compounds, (ii) a C4 leaf spiked with C3 starch, and (iii) two natural plant samples (root, leaf). Starch preparation methods based on enzymatic or acid hydrolysis did not yield similar results and exhibited contaminations by non-starch compounds. The specificity of the acidic hydrolysis method was especially low, and we therefore suggest terming these preparations as HCl-hydrolysable carbon, rather than starch. Despite being more specific, enzyme-based methods to isolate starch also need to be further optimized to increase specificity. The analysis of sugars by ion-exchange methods (bulk preparations) was fast but produced more variable isotope compositions than compound-specific methods. Compound-specific approaches did not in all cases correctly reproduce the target values, mainly due to unsatisfactory separation of sugars and background contamination. Our study demonstrates that, despite their wide application, methods for the preparation of starch and soluble sugars for the analysis of carbon isotope composition are not (yet) reliable enough to be routinely applied and further research is urgently needed to resolve the identified problems. Copyright © 2009 John Wiley & Sons, Ltd.