Metabolism is a major driver of hydrogen isotope fractionation recorded in tree-ring glucose of Pinus nigra.

Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and biogeosciences. To address these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree-ring series (1961-1995). We found fractionation signals (i.e. temporal variability in deuterium abundance) at glucose H1 and H2 introduced by closely related metabolic processes. Regression analysis indicates that these signals (and thus metabolism) respond to drought and atmospheric CO2 concentration beyond a response change point. They explain ≈ 60% of the whole-molecule deuterium variability. Altered metabolism is associated with below-average yet not exceptionally low growth. We propose the signals are introduced at the leaf level by changes in sucrose-to-starch carbon partitioning and anaplerotic carbon flux into the Calvin-Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose.

Detecting long-term metabolic shifts using isotopomers: CO2-driven suppression of photorespiration in C3 plants over the 20th century.

Terrestrial vegetation currently absorbs approximately a third of anthropogenic CO2 emissions, mitigating the rise of atmospheric CO2. However, terrestrial net primary production is highly sensitive to atmospheric CO2 levels and associated climatic changes. In C3 plants, which dominate terrestrial vegetation, net photosynthesis depends on the ratio between photorespiration and gross photosynthesis. This metabolic flux ratio depends strongly on CO2 levels, but changes in this ratio over the past CO2 rise have not been analyzed experimentally. Combining CO2 manipulation experiments and deuterium NMR, we first establish that the intramolecular deuterium distribution (deuterium isotopomers) of photosynthetic C3 glucose contains a signal of the photorespiration/photosynthesis ratio. By tracing this isotopomer signal in herbarium samples of natural C3 vascular plant species, crops, and a Sphagnum moss species, we detect a consistent reduction in the photorespiration/photosynthesis ratio in response to the ∼100-ppm CO2 increase between ∼1900 and 2013. No difference was detected in the isotopomer trends between beet sugar samples covering the 20th century and CO2 manipulation experiments, suggesting that photosynthetic metabolism in sugar beet has not acclimated to increasing CO2 over >100 y. This provides observational evidence that the reduction of the photorespiration/photosynthesis ratio was ca. 25%. The Sphagnum results are consistent with the observed positive correlations between peat accumulation rates and photosynthetic rates over the Northern Hemisphere. Our results establish that isotopomers of plant archives contain metabolic information covering centuries. Our data provide direct quantitative information on the "CO2 fertilization" effect over decades, thus addressing a major uncertainty in Earth system models.

Compound-specific carbon, nitrogen, and hydrogen isotope analysis of N-nitrosodimethylamine in aqueous solutions.

Mitigation of N-nitrosodimethylamine (NDMA) and other hazardous water disinfection byproducts (DBP) is currently hampered by a limited understanding of DBP formation mechanisms. Because variations of the stable isotope composition of NDMA can potentially reveal reaction pathways and precursor compounds, we developed a method for the compound-specific isotope analysis (CSIA) of (13)C/(12)C, (15)N/(14)N, and (2)H/(1)H ratios of NDMA by gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). Method quantification limits for the accurate isotope analysis of NDMA, N-nitrosodiethyl-, -dipropyl-, and -dibutylamine as well as N-nitrosopyrrolidine were between 0.18 to 0.60 nmol C, 0.40 to 0.80 nmol N, and 2.2 to 5.8 nmol H injected on column. Coupling solid phase extraction (SPE) to GC/IRMS enabled the precise quantification of C, N, and H isotope ratios of NDMA in aqueous samples at concentrations of 0.6 µM (45 µg L(-1)). We validated the proposed method with a laboratory experiment, in which NDMA was formed with stoichiometric yield (97 ± 4%) through chloramination of the pharmaceutical ranitidine (3 µM). δ(13)C and δ(2)H values of NDMA remained constant during NDMA formation while its δ(15)N increased due to a reaction at a N atom in the rate-limiting step of NDMA formation. The δ(2)H value of NDMA determined by SPE-GC/IRMS also corresponded well to the δ(2)H value of the N(CH3)2-group of ranitidine measured by quantitative deuterium nuclear magnetic resonance spectroscopy. This observation implies that the N(CH3)2-moiety of ranitidine is transferred to NDMA without being chemically altered and illustrates the accuracy of the proposed method.

Global CO2 fertilization of Sphagnum peat mosses via suppression of photorespiration during the twentieth century.

Natural peatlands contribute significantly to global carbon sequestration and storage of biomass, most of which derives from Sphagnum peat mosses. Atmospheric CO2 levels have increased dramatically during the twentieth century, from 280 to > 400 ppm, which has affected plant carbon dynamics. Net carbon assimilation is strongly reduced by photorespiration, a process that depends on the CO2 to O2 ratio. Here we investigate the response of the photorespiration to photosynthesis ratio in Sphagnum mosses to recent CO2 increases by comparing deuterium isotopomers of historical and contemporary Sphagnum tissues collected from 36 peat cores from five continents. Rising CO2 levels generally suppressed photorespiration relative to photosynthesis but the magnitude of suppression depended on the current water table depth. By estimating the changes in water table depth, temperature, and precipitation during the twentieth century, we excluded potential effects of these climate parameters on the observed isotopomer responses. Further, we showed that the photorespiration to photosynthesis ratio varied between Sphagnum subgenera, indicating differences in their photosynthetic capacity. The global suppression of photorespiration in Sphagnum suggests an increased net primary production potential in response to the ongoing rise in atmospheric CO2, in particular for mire structures with intermediate water table depths.

Intramolecular 13C analysis of tree rings provides multiple plant ecophysiology signals covering decades.

Measurements of carbon isotope contents of plant organic matter provide important information in diverse fields such as plant breeding, ecophysiology, biogeochemistry and paleoclimatology. They are currently based on 13C/12C ratios of specific, whole metabolites, but we show here that intramolecular ratios provide higher resolution information. In the glucose units of tree-ring cellulose of 12 tree species, we detected large differences in 13C/12C ratios (>10‰) among carbon atoms, which provide isotopically distinct inputs to major global C pools, including wood and soil organic matter. Thus, considering position-specific differences can improve characterisation of soil-to-atmosphere carbon fluxes and soil metabolism. In a Pinus nigra tree-ring archive formed from 1961 to 1995, we found novel 13C signals, and show that intramolecular analysis enables more comprehensive and precise signal extraction from tree rings, and thus higher resolution reconstruction of plants' responses to climate change. Moreover, we propose an ecophysiological mechanism for the introduction of a 13C signal, which links an environmental shift to the triggered metabolic shift and its intramolecular 13C signature. In conclusion, intramolecular 13C analyses can provide valuable new information about long-term metabolic dynamics for numerous applications.

Elucidating turnover pathways of bioactive small molecules by isotopomer analysis: the persistent organic pollutant DDT.

The persistent organic pollutant DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane) is still indispensable in the fight against malaria, although DDT and related compounds pose toxicological hazards. Technical DDT contains the dichloro congener DDD (1-chloro-4-[2,2-dichloro-1-(4-chlorophenyl)ethyl]benzene) as by-product, but DDD is also formed by reductive degradation of DDT in the environment. To differentiate between DDD formation pathways, we applied deuterium NMR spectroscopy to measure intramolecular deuterium distributions (2H isotopomer abundances) of DDT and DDD. DDD formed in the technical DDT synthesis was strongly deuterium-enriched at one intramolecular position, which we traced back to 2H/1H fractionation of a chlorination step in the technical synthesis. In contrast, DDD formed by reductive degradation was strongly depleted at the same position, which was due to the incorporation of 2H-depleted hydride equivalents during reductive degradation. Thus, intramolecular isotope distributions give mechanistic information on reaction pathways, and explain a puzzling difference in the whole-molecule 2H/1H ratio between DDT and DDD. In general, our results highlight that intramolecular isotope distributions are essential to interpret whole-molecule isotope ratios. Intramolecular isotope information allows distinguishing pathways of DDD formation, which is important to identify polluters or to assess DDT turnover in the environment. Because intramolecular isotope data directly reflect isotope fractionation of individual chemical reactions, they are broadly applicable to elucidate transformation pathways of small bioactive molecules in chemistry, physiology and environmental science.

Two-dimensional ³¹P,¹H NMR spectroscopic profiling of phospholipids in cheese and fish.

Phospholipids (PLs) comprise an important lipid class in food because of their technological use as emulsifiers and their nutritional value. This study used one-dimensional (31)P NMR and two-dimensional (2D) (31)P,(1)H COSY NMR spectroscopy for the determination of the PL composition of cheese and fish after liquid-liquid enrichment. This extraction step enabled the identification of 10 PLs in cheese and 9 PLs in fish by 2D (31)P,(1)H NMR. Variations in the (31)P shifts indicated differences in the fatty acids attached to the individual PLs. The total PL content in cheese fat and fish oil ranged from 0.3 to 0.4% and from 5 to 12%, respectively. Phosphatidylcholine was the most prominent PL in both matrices (up to 65%). Minor PLs (limit of detection = 4 nmol, i.e. 500 µL of an 8 µM solution) were identified in forms of phosphatidic acid, lysophosphatidic acid, and phosphatidylglycerol. Specific cross couplings and (1)H fine structures in the 2D (31)P,(1)H NMR spectra proved to be valuable for the assignment and verification of known and uncommon PLs in the samples.