Comparison of Carbon Isotope Ratio Measurement of the Vanillin Methoxy Group by GC-IRMS and 13C-qNMR.

Site-specific carbon isotope ratio measurements by quantitative 13C NMR (13C-qNMR), Orbitrap-MS, and GC-IRMS offer a new dimension to conventional bulk carbon isotope ratio measurements used in food provenance, forensics, and a number of other applications. While the site-specific measurements of carbon isotope ratios in vanillin by 13C-qNMR or Orbitrap-MS are powerful new tools in food analysis, there are a limited number of studies regarding the validity of these measurement results. Here we present carbon site-specific measurements of vanillin by GC-IRMS and 13C-qNMR for methoxy carbon. Carbon isotope delta (δ13C) values obtained by these different measurement approaches demonstrate remarkable agreement; in five vanillin samples whose bulk δ13C values ranged from -31‰ to -26‰, their δ13C values of the methoxy carbon ranged from -62.4‰ to -30.6‰, yet the difference between the results of the two analytical approaches was within ±0.6‰. While the GC-IRMS approach afforded up to 9-fold lower uncertainties and required 100-fold less sample compared to the 13C-qNMR, the 13C-qNMR is able to assign δ13C values to all carbon atoms in the molecule, not just the cleavable methoxy group.

Natural Abiotic Iron-Oxido-Mediated Formation of C1 and C2 Compounds from Environmentally Important Methyl-Substituted Substrates.

Organic and inorganic volatile compounds containing one carbon atom (C1), such as carbon dioxide, methane, methanol, formaldehyde, carbon monoxide, and chloromethane, are ubiquitous in the environment, are key components in global carbon cycling, play an important role in atmospheric physics and chemistry, e.g., as greenhouse gases, destroy stratospheric and tropospheric ozone, and control the atmospheric oxidation capacity. Up to now, most C1 compounds in the environment were associated with complex metabolic and enzymatic pathways in organisms or to combustion processes of organic matter. We now present compelling evidence that many C1 and C2 compounds have a common origin in methyl groups of methyl-substituted substrates that are cleaved by the iron oxide-mediated formation of methyl radicals. This scenario is derived from experiments with a mechanistically well-studied bispidine-iron-oxido complex as oxidant and dimethyl sulfoxide as the environmentally relevant model substrate and is supported by computational modeling based on density functional theory and ab initio quantum-chemical studies. The exhaustive experimental model studies, also involving extensive isotope labeling, are complemented with the substitution of the bispidine model system by environmentally relevant iron oxides and, finally, a collection of soils with varying iron and organic matter contents. The combination of all data suggests that the iron oxide-mediated formation of methyl radicals from methyl-substituted substrates is a common abiotic source for widespread C1 and C2 compounds in the environment.

Methane formation driven by light and heat prior to the origin of life and beyond.

Methane is a potent greenhouse gas, which likely enabled the evolution of life by keeping the early Earth warm. Here, we demonstrate routes towards abiotic methane and ethane formation under early-earth conditions from methylated sulfur and nitrogen compounds with prebiotic origin. These compounds are demethylated in Fenton reactions governed by ferrous iron and reactive oxygen species (ROS) produced by light and heat in aqueous environments. After the emergence of life, this phenomenon would have greatly intensified in the anoxic Archean by providing methylated sulfur and nitrogen substrates. This ROS-driven Fenton chemistry can occur delocalized from serpentinization across Earth's humid realm and thereby substantially differs from previously suggested methane formation routes that are spatially restricted. Here, we report that Fenton reactions driven by light and heat release methane and ethane and might have shaped the chemical evolution of the atmosphere prior to the origin of life and beyond.

Methane accumulation and its potential precursor compounds in the oxic surface water layer of two contrasting stratified lakes.

Methane (CH4) supersaturation in oxygenated waters is a widespread phenomenon despite the traditional perception of strict anoxic methanogenesis. This notion has recently been challenged by successive findings of processes and mechanisms that produce CH4 in oxic environments. While some of the processes contributing to the vertical accumulation of CH4 in the oxygenated upper water layers of freshwater lakes have been identified, temporal variations as well as drivers are still poorly understood. In this study, we investigated the accumulation of CH4 in oxic water layers of two contrasting lakes in Germany: Lake Willersinnweiher (shallow, monomictic, eutrophic) and Lake Stechlin (deep, dimictic, eutrophic) from 2019 to 2020. The dynamics of isotopic values of CH4 and the role of potential precursor compounds of oxic CH4 production were explored. During the study period, persistent strong CH4 supersaturation (relative to air) was observed in the surface waters, mostly concentrated around the thermocline. The magnitude of vertical CH4 accumulation strongly varied over season and was generally more pronounced in shallow Lake Willersinnweiher. In both lakes, increases in CH4 concentrations from the surface to the thermocline mostly coincided with an enrichment in 13C-CH4 and 2H-CH4, indicating a complex interaction of multiple processes such as CH4 oxidation, CH4 transport from littoral sediments and oxic CH4 production, sustaining and controlling this CH4 supersaturation. Furthermore, incubation experiments with 13C- and 2H-labelled methylated P-, N- and C- compounds clearly showed that methylphosphonate, methylamine and methionine acted as potent precursors of accumulating CH4 and at least partly sustained CH4 supersaturation. This highlights the need to better understand the mechanisms underlying CH4 accumulation by focusing on production and transport pathways of CH4 and its precursor compounds, e.g., produced via phytoplankton. Such knowledge forms the foundation to better predict aquatic CH4 dynamics and its subsequent rates of emission to the atmosphere.

Effect of immune responses on breath methane dynamics.

Methane (CH4) which can be detected in human breath has long been exclusively associated with anaerobic microbial activity (methanogenesis) in the gastrointestinal tract. However, recent studies challenge this understanding by revealing that CH4might also be produced endogenously in cells through oxidative-reductive stress reactions. Consequently, variations in breath CH4levels compared to an individual's baseline level might indicate enhanced oxidative stress levels, and, therefore, monitoring breath CH4levels might offer great potential for 'in vivo' diagnostics such as disease diagnosis, monitoring the efficacy of treatments, or during the application of personalized medicine. To evaluate the effects from immune responses triggered by infections, inflammations, and induced perturbation by vaccination on CH4dynamics in breath, two subjects were monitored over a period of almost 2 years. Breath CH4levels were measured by gas chromatography equipped with a flame-ionization detector. Both subjects exhibited significant deviations (positive and negative, respectively) from their normal CH4breath levels during periods of potential enhanced immune activity. Deviations from the 'healthy state' were indicated by the exceeding of individual CH4ranges. Moreover, for the first time we could clearly prove CH4degradation induced through vaccination by measuring stable carbon isotopes of CH4using gas chromatograph-combustion-isotope ratio mass spectrometry. Hence, breath CH4concentration and isotopic analyses may be used as a biomarker to evaluate specific immune responses and individual immune states.

Radical-Driven Methane Formation in Humans Evidenced by Exogenous Isotope-Labeled DMSO and Methionine.

Methane (CH4), which is produced endogenously in animals and plants, was recently suggested to play a role in cellular physiology, potentially influencing the signaling pathways and regulatory mechanisms involved in nitrosative and oxidative stress responses. In addition, it was proposed that the supplementation of CH4 to organisms may be beneficial for the treatment of several diseases, including ischemia, reperfusion injury, and inflammation. However, it is still unclear whether and how CH4 is produced in mammalian cells without the help of microorganisms, and how CH4 might be involved in physiological processes in humans. In this study, we produced the first evidence of the principle that CH4 is formed non-microbially in the human body by applying isotopically labeled methylated sulfur compounds, such as dimethyl sulfoxide (DMSO) and methionine, as carbon precursors to confirm cellular CH4 formation. A volunteer applied isotopically labeled (2H and 13C) DMSO on the skin, orally, and to blood samples. The monitoring of stable isotope values of CH4 convincingly showed the conversion of the methyl groups, as isotopically labeled CH4 was formed during all experiments. Based on these results, we considered several hypotheses about endogenously formed CH4 in humans, including physiological aspects and stress responses involving reactive oxygen species (ROS). While further and broader validation studies are needed, the results may unambiguously serve as a proof of concept for the endogenous formation of CH4 in humans via a radical-driven process. Furthermore, these results might encourage follow-up studies to decipher the potential physiological role of CH4 and its bioactivity in humans in more detail. Of particular importance is the potential to monitor CH4 as an oxidative stress biomarker if the observed large variability of CH4 in breath air is an indicator of physiological stress responses and immune reactions. Finally, the potential role of DMSO as a radical scavenger to counteract oxidative stress caused by ROS might be considered in the health sciences. DMSO has already been investigated for many years, but its potential positive role in medical use remains highly uncertain.

Mid- to late Holocene sea-level rise recorded in Hells Bells 234U/238U ratio and geochemical composition.

Hells Bells are underwater secondary carbonates discovered in sinkholes (cenotes) southeast of Cancun on the north-eastern Yucatán peninsula, Mexico. These authigenic calcite precipitates, reaching a length of up to 4 m, most likely grow in the pelagic redoxcline. Here we report on detailed 230Th/U-dating and in-depth geochemical and stable isotope analyses of specimens from cenotes El Zapote, Maravilla and Tortugas. Hells Bells developed since at least ~ 8000 years ago, with active growth until present day. Initial (234U/238U) activity ratios (δ234U0) in Hells Bells calcite decreas from 55 to 15‰ as sea level converges toward its present state. The temporal evolution of the geochemistry and isotope composition of Hells Bells calcites thus appears to be closely linked to sea-level rise and reflects changing hydrological conditions (desalinization) of the aquifer. We suggest that decelerated leaching of excess 234U from the previously unsaturated bedrock traces Holocene relative sea-level rise. Considering this proxy, the resulting mean sea-level reconstruction contains half as much scatter, i.e. improves by a factor of two, when compared to previously published work for the period between 8 and 4 ky BP.

ROS-driven cellular methane formation: Potential implications for health sciences.

Recently it has been proposed that methane might be produced by all living organisms via a mechanism driven by reactive oxygen species that arise through the metabolic activity of cells. Here, we summarise details of this novel reaction pathway and discuss its potential significance for clinical and health sciences. In particular, we highlight the role of oxidative stress in cellular methane formation. As several recent studies also demonstrated the anti-inflammatory potential for exogenous methane-based approaches in mammalians, this article addresses the intriguing question if ROS-driven methane formation has a general physiological role and associated diagnostic potential.

Methane formation driven by reactive oxygen species across all living organisms.

Methane (CH4), the most abundant hydrocarbon in the atmosphere, originates largely from biogenic sources1 linked to an increasing number of organisms occurring in oxic and anoxic environments. Traditionally, biogenic CH4 has been regarded as the final product of anoxic decomposition of organic matter by methanogenic archaea. However, plants2,3, fungi4, algae5 and cyanobacteria6 can produce CH4 in the presence of oxygen. Although methanogens are known to produce CH4 enzymatically during anaerobic energy metabolism7, the requirements and pathways for CH4 production by non-methanogenic cells are poorly understood. Here, we demonstrate that CH4 formation by Bacillus subtilis and Escherichia coli is triggered by free iron and reactive oxygen species (ROS), which are generated by metabolic activity and enhanced by oxidative stress. ROS-induced methyl radicals, which are derived from organic compounds containing sulfur- or nitrogen-bonded methyl groups, are key intermediates that ultimately lead to CH4 production. We further show CH4 production by many other model organisms from the Bacteria, Archaea and Eukarya domains, including in several human cell lines. All these organisms respond to inducers of oxidative stress by enhanced CH4 formation. Our results imply that all living cells probably possess a common mechanism of CH4 formation that is based on interactions among ROS, iron and methyl donors, opening new perspectives for understanding biochemical CH4 formation and cycling.

Making plant methane formation visible-Insights from application of 13C-labeled dimethyl sulfoxide.

Methane (CH4) formation by vegetation has been studied intensively over the last 15 years. However, reported CH4 emissions vary by several orders of magnitude, thus making global estimates difficult. Moreover, the mechanism(s) for CH4 formation by plants is (are) largely unknown.Here, we introduce a new approach for making CH4 formation by plants clearly visible. By application of 13C-labeled dimethyl sulfoxide (DMSO) onto the leaves of tobacco plants (Nicotiana tabacum) and Chinese silver grass (Miscanthus sinensis) the effect of light and dark conditions on CH4 formation of this pathway was examined by monitoring stable carbon isotope ratios of headspace CH4 (δ13C-CH4 values).Both plant species showed increasing headspace δ13C-CH4 values while exposed to light. Higher light intensities increased CH4 formation rates in N. tabacum but decreased rates for M. sinensis. In the dark no formation of CH4 could be detected for N. tabacum, while M. sinensis still produced ~50% of CH4 compared to that during light exposure.Our findings suggest that CH4 formation is clearly dependent on light conditions and plant species and thus indicate that DMSO is a potential precursor of vegetative CH4. The novel isotope approach has great potential to investigate, at high temporal resolution, physiological, and environmental factors that control pathway-specific CH4 emissions from plants.

A surprise from the deep.

Isotope patterns of methoxyl groups reveal the origin an d production potential of methane from coal.

13 C-chloromethane incubations provide evidence for novel bacterial chloromethane degraders in a living tree fern.

Chloromethane (CH3 Cl) is the most abundant halogenated volatile organic compound in the atmosphere and contributes to stratospheric ozone depletion. CH3 Cl has mainly natural sources such as emissions from vegetation. In particular, ferns have been recognized as strong emitters. Mitigation of CH3 Cl to the atmosphere by methylotrophic bacteria, a global sink for this compound, is likely underestimated and remains poorly characterized. We identified and characterized CH3 Cl-degrading bacteria associated with intact and living tree fern plants of the species Cyathea australis by stable isotope probing (SIP) with 13 C-labelled CH3 Cl combined with metagenomics. Metagenome-assembled genomes (MAGs) related to Methylobacterium and Friedmanniella were identified as being involved in the degradation of CH3 Cl in the phyllosphere, i.e., the aerial parts of the tree fern, while a MAG related to Sorangium was linked to CH3 Cl degradation in the fern rhizosphere. The only known metabolic pathway for CH3 Cl degradation, via a methyltransferase system including the gene cmuA, was not detected in metagenomes or MAGs identified by SIP. Hence, a yet uncharacterized methylotrophic cmuA-independent pathway may drive CH3 Cl degradation in the investigated tree ferns.

Temperature signal recorded in δ2H and δ13C values of wood lignin methoxyl groups from a permafrost forest in northeastern China.

Stable isotopes in wood lignin methoxyl groups (δ2HLM and δ13CLM values) have been suggested as valuable complementary paleoclimate proxies. In permafrost forests, tree growth is influenced by multiple factors, however temperature appears to have the strongest impact on tree growth and, therefore, on carbon cycling. To test whether δ2HLM and δ13CLM values of trees from permafrost regions might record climate parameters, two dominant tree species (Larix gmelinii, larch, and Pinus sylvestris var. mongolica, pine) collected from a permafrost forest in China's Greater Hinggan Mountains, were investigated. The two tree species larch and pine covered time spans of 1940 to 2013 and 1870 to 2013, respectively. Results showed significant correlations of pine and larch δ2HLM values and larch δ13CLM values with temperatures and in particular with the mean temperature of the growing season from April to August. However, only weak correlations of δ2HLM and δ13CLM values with moisture conditions, such as precipitation amount and relative humidity were observed. In addition, species specificity in the climate response was most obvious for δ13CLM values. Compared to a temperature reconstruction based on tree ring width, pine δ2HLM-based reconstruction showed strongest spatial correlations with regional temperature. Therefore, δ2HLM values might be a promising proxy to reconstruct growing-season temperatures in permafrost regions.

Sources and sinks of chloromethane in a salt marsh ecosystem: constraints from concentration and stable isotope measurements of laboratory incubation experiments.

Chloromethane (CH3Cl) is the most abundant long-lived chlorinated organic compound in the atmosphere and contributes significantly to natural stratospheric ozone depletion. Salt marsh ecosystems including halophyte plants are a known source of atmospheric CH3Cl but estimates of their total global source strength are highly uncertain and knowledge of the major production and consumption processes in the atmosphere-halophyte-soil system is yet incomplete. In this study we investigated the halophyte plant, Salicornia europaea, and soil samples from a coastal salt marsh site in Sardinia/Italy for their potential to emit and consume CH3Cl and using flux measurements, stable isotope techniques and Arrhenius plots differentiated between biotic and abiotic processes. Our laboratory approach clearly shows that at least 6 different production and consumption processes are active in controlling atmospheric CH3Cl fluxes of a salt marsh ecosystem. CH3Cl release by dried plant and soil material was substantially higher than that from the fresh material at temperatures ranging from 20 to 70 °C. Results of Arrhenius plots helped to distinguish between biotic and abiotic formation processes in plants and soils. Biotic CH3Cl consumption rates were highest at 30 °C for plants and 50 °C for soils, and microbial uptake was higher in soils with higher organic matter content. Stable isotope techniques helped to distinguish between formation and degradation processes and also provided a deeper insight into potential methyl moiety donor compounds, such as S-adenosyl-l-methionine, S-methylmethionine and pectin, that might be involved in the abiotic and biotic CH3Cl production processes. Our results clearly indicate that cycling of CH3Cl in salt marsh ecosystems is a result of several biotic and abiotic processes occurring simultaneously in the atmosphere-plant-soil system. Important precursor compounds for biotic and abiotic CH3Cl formation might be methionine derivatives and pectin. All formation and degradation processes are temperature dependent and thus environmental changes might affect the strength of each source and sink within salt marsh ecosystems and thus considerably alter total fluxes of CH3Cl from salt marsh ecosystems to the atmosphere.

High Spatiotemporal Dynamics of Methane Production and Emission in Oxic Surface Water.

The discovery of methane (CH4) accumulation in oxic marine and limnic waters has redefined the role of aquatic environments in the regional CH4 cycle. Although CH4 accumulation in oxic surface waters became apparent in recent years, the sources are still subject to controversial discussions. We present high-resolution in situ measurements of CH4 concentration and its stable isotope composition in a stratified mesotrophic lake. We show that CH4 accumulation in surface waters originates from a highly dynamic interplay between (oxic) CH4 production and emission to the atmosphere. Laboratory incubations of different phytoplankton types and application of stable isotope techniques provide a first unambiguous evidence that major phytoplankton classes in Lake Stechlin per se produce CH4 under oxic conditions. Combined field and lab results show that the photoautotroph community is an important driver for CH4 production and its highly dynamic accumulation in oxic surface waters.

Chlorine Isotope Fractionation of the Major Chloromethane Degradation Processes in the Environment.

Chloromethane (CH3Cl) is an important source of chlorine in the stratosphere, but detailed knowledge of the magnitude of its sources and sinks is missing. Here, we measured the stable chlorine isotope fractionation (εCl) associated with the major abiotic and biotic CH3Cl sinks in the environment, namely, CH3Cl degradation by hydroxyl (·OH) and chlorine (·Cl) radicals in the troposphere and by reference bacteria Methylorubrum extorquens CM4 and Leisingera methylohalidivorans MB2 from terrestrial and marine environments, respectively. No chlorine isotope fractionation was detected for reaction of CH3Cl with ·OH and ·Cl radicals, whereas a large chlorine isotope fractionation (εCl) of -10.9 ± 0.7‰ (n = 3) and -9.4 ± 0.9 (n = 3) was found for CH3Cl degradation by M. extorquens CM4 and L. methylohalidivorans MB2, respectively. The large difference in chlorine isotope fractionation observed between tropospheric and bacterial degradation of CH3Cl provides an effective isotopic tool to characterize and distinguish between major abiotic and biotic processes contributing to the CH3Cl sink in the environment. Our findings demonstrate the potential of emerging triple-element isotopic approaches including chlorine to carbon and hydrogen analysis for the assessment of global cycling of organochlorines.

Methane Production and Bioactivity-A Link to Oxido-Reductive Stress.

Biological methane formation is associated with anoxic environments and the activity of anaerobic prokaryotes (Archaea). However, recent studies have confirmed methane release from eukaryotes, including plants, fungi, and animals, even in the absence of microbes and in the presence of oxygen. Furthermore, it was found that aerobic methane emission in plants is stimulated by a variety of environmental stress factors, leading to reactive oxygen species (ROS) generation. Further research presented evidence that molecules with sulfur and nitrogen bonded methyl groups such as methionine or choline are carbon precursors of aerobic methane formation. Once generated, methane is widely considered to be physiologically inert in eukaryotes, but several studies have found association between mammalian methanogenesis and gastrointestinal (GI) motility changes. In addition, a number of recent reports demonstrated anti-inflammatory potential for exogenous methane-based approaches in model anoxia-reoxygenation experiments. It has also been convincingly demonstrated that methane can influence the downstream effectors of transiently increased ROS levels, including mitochondria-related pro-apoptotic pathways during ischemia-reperfusion (IR) conditions. Besides, exogenous methane can modify the outcome of gasotransmitter-mediated events in plants, and it appears that similar mechanism might be active in mammals as well. This review summarizes the relevant literature on methane-producing processes in eukaryotes, and the available results that underscore its bioactivity. The current evidences suggest that methane liberation and biological effectiveness are both linked to cellular redox regulation. The data collectively imply that exogenous methane influences the regulatory mechanisms and signaling pathways involved in oxidative and nitrosative stress responses, which suggests a modulator role for methane in hypoxia-linked pathologies.

Methylotrophs and Methylotroph Populations for Chloromethane Degradation.

Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to the stratosphere, its photolysis contributes to the degradation of stratospheric ozone. A better knowledge of chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of chloromethane by methylotrophic communities in terrestrial environments is a major underestimated chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using chloromethane as the sole carbon source. In addition to anaerobes that degrade chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use chloromethane as sole carbon and energy source. Among those, the Proteobacterium Methylobacterium (recently reclassified as Methylorubrum) harbours the only characterisized 'chloromethane utilization' (cmu) pathway, so far. This pathway is not representative of chloromethane-utilizing populations in the environment as cmu genes are rare in metagenomes. Recently, combined 'omics' biological approaches with chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated chloromethane-degraders lacking the cmu genes display a singular isotope fractionation signature of chloromethane. Moreover, 13CH3Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from the taxa known for chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global chloromethane cycle.

Methyl sulfates as methoxy isotopic reference materials for δ13 C and δ2 H measurements.

RATIONALE: Stable hydrogen and carbon isotope ratios of methoxy groups (OCH3 ) of plant organic matter have many potential applications in biogeochemical, atmospheric and food research. So far, most of the analyses of plant methoxy groups by isotope ratio mass spectrometry have employed liquid iodomethane (CH3 I) as the reference material to normalise stable isotope measurements of these moieties to isotope-δ scales. However, comparisons of measurements of stable hydrogen and carbon isotopes of plant methoxy groups are still hindered by the lack of suitable reference materials. METHODS: We have investigated two methyl sulfate salts (HUBG1 and HUBG2), which exclusively contain carbon and hydrogen from one methoxy group, for their suitability as methoxy reference materials. Firstly, the stable hydrogen and carbon isotope values of the bulk compounds were calibrated against international reference substances by high-temperature conversion- and elemental analyser isotope ratio mass spectrometry (HTC- and EA-IRMS). In a second step these values were compared with values obtained by measurements using gas chromatography/isotope ratio mass spectrometry (GC/IRMS) where prior to analysis the methoxy groups were converted into gaseous iodomethane. RESULTS: The 2 H- and 13 C isotopic abundances of HUBG1 measured by HTC- and EA-IRMS and expressed as δ-values on the usual international scales are -144.5 ± 1.2 mUr (n = 30) and -50.31 ± 0.16 mUr (n = 14), respectively. For HUBG2 we obtained -102.0 ± 1.3 mUr (n = 32) and +1.60 ± 0.12 mUr (n = 16). Furthermore, the values obtained by GC/IRMS were in good agreement with the HTC- and EA-IRMS values. CONCLUSIONS: We suggest that both methyl sulfates are suitable reference materials for normalisation of isotope measurements of carbon of plant methoxy groups to isotope-δ scales and for inter-laboratory calibration. For stable hydrogen isotope measurements, we suggest that in addition to HUBG1 and HUBG2 additional reference materials are required to cover the full range of plant methoxy groups reported so far.