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Traditional leaf gas exchange experiments have focused on net CO2 exchange (Anet). Here, using California poplar (Populus trichocarpa), we coupled measurements of net oxygen production (NOP), isoprene emissions and δ18O in O2 to traditional CO2/H2O gas exchange with chlorophyll fluorescence, and measured light, CO2 and temperature response curves. This allowed us to obtain a comprehensive picture of the photosynthetic redox budget including electron transport rate (ETR) and estimates of the mean assimilatory quotient (AQ = Anet/NOP). We found that Anet and NOP were linearly correlated across environmental gradients with similar observed AQ values during light (1.25 ± 0.05) and CO2 responses (1.23 ± 0.07). In contrast, AQ was suppressed during leaf temperature responses in the light (0.87 ± 0.28), potentially due to the acceleration of alternative ETR sinks like lipid synthesis. Anet and NOP had an optimum temperature (Topt) of 31°C, while ETR and δ18O in O2 (35°C) and isoprene emissions (39°C) had distinctly higher Topt. The results confirm a tight connection between water oxidation and ETR and support a view of light-dependent lipid synthesis primarily driven by photosynthetic ATP/NADPH not consumed by the Calvin-Benson cycle, as an important thermotolerance mechanism linked with high rates of (photo)respiration and CO2/O2 recycling.

Stem respiration is a quantitatively important, but poorly understood component of ecosystem carbon cycling in terrestrial ecosystems. However, a dynamic stem gas exchange system for quantifying real-time stem carbon dioxide (CO2) efflux (Es) is not commercially available resulting in limited observations based on the static method where air is recirculated through a stem enclosure. The static method has limited temporal resolution, suffers from condensation issues, requires a leak-free enclosure, which is often difficult to verify in the field, and requires physically removing the chamber or flushing it with ambient air before starting each measurement.•With the goal of improving our quantitative understanding of biophysical, physiological, biochemical, and environmental factors that influence diurnal Es patterns, here we present a custom system for quantifying real-time stem Es in remote tropical forests.•The system is low cost, lightweight, and waterproof with low power requirements (1.2-2.4 W) for real-time monitoring of stem Es using a 3D printed dynamic stem chamber and a 12V car battery. The design offers control over the flow rate through the stem chamber, eliminates the need for a pump to introduce air into the chamber, and water condensation issues by removing water vapor prior to CO2 analysis.•Following a simple CO2 infrared gas analyzer (IRGA) calibration and match procedure with a 400-ppm standard, we quantified diurnal Es observations over a 24-hours period during the summer growing season from an ash tree (Fraxinus sp.) in Fort Collins, Colorado. The results are consistent with previous laboratory and field studies that show Es can be suppressed during the day relative to the night.

Growth suppression and defence signalling are simultaneous strategies that plants invoke to respond to abiotic stress. Here, we show that the drought stress response of poplar trees (Populus trichocarpa) is initiated by a suppression in cell wall derived methanol (MeOH) emissions and activation of acetic acid (AA) fermentation defences. Temperature sensitive emissions dominated by MeOH (AA/MeOH

Although apparent light inhibition of leaf day respiration is a widespread reported phenomenon, the mechanisms involved, including utilization of alternate respiratory pathways and substrates and light inhibition of TCA cycle enzymes are under active investigation. Recently, acetate fermentation was highlighted as a key drought survival strategy mediated through protein acetylation and jasmonate signaling. Here, we evaluate the light-dependence of acetate transport and assimilation in Populus trichocarpa trees using the dynamic xylem solution injection (DXSI) method developed here for continuous studies of C1 and C2 organic acid transport and light-dependent metabolism. Over 7 days, 1.0 L of [13C]formate and [13C2]acetate solutions were delivered to the stem base of 2-year old potted poplar trees, while continuous diurnal observations were made in the canopy of CO2, H2O, and isoprene gas exchange together with δ13CO2. Stem base injection of 10 mM [13C2]acetate induced an overall pattern of canopy branch headspace 13CO2 enrichment (δ13CO2 +27‱) with a diurnal structure in δ13CO2 reaching a mid-day minimum followed by a maximum shortly after darkening where δ13CO2 values rapidly increased up to +12‱. In contrast, 50 mM injections of [13C]formate were required to reach similar δ13CO2 enrichment levels in the canopy with δ13CO2 following diurnal patterns of transpiration. Illuminated leaves of detached poplar branches pretreated with 10 mM [13C2]acetate showed lower δ13CO2 (+20‱) compared to leaves treated with 10 mM [13C]formate (+320‱), the opposite pattern observed at the whole plant scale. Following dark/light cycles at the leaf-scale, rapid, strong, and reversible enhancements in headspace δ13CO2 by up to +60‱ were observed in [13C2]acetate-treated leaves which showed enhanced dihydrojasmonic acid and TCA cycle intermediate concentrations. The results are consistent with acetate in the transpiration stream as an effective activator of the jasmonate signaling pathway and respiratory substrate. The shorter lifetime of formate relative to acetate in the transpiration stream suggests rapid formate oxidation to CO2 during transport to the canopy. In contrast, acetate is efficiently transported to the canopy where an increased allocation towards mitochondrial dark respiration occurs at night. The results highlight the potential for an effective integration of acetate into glyoxylate and TCA cycles and the light-inhibition of citrate synthase as a potential regulatory mechanism controlling the diurnal allocation of acetate between anabolic and catabolic processes.

Although primarily studied in relation to photorespiration, serine metabolism in chloroplasts may play a key role in plant CO2 fertilization responses by linking CO2 assimilation with growth. Here, we show that the phosphorylated serine pathway is part of a 'photosynthetic C1 pathway' and demonstrate its high activity in foliage of a C3 tree where it rapidly integrates photosynthesis and C1 metabolism contributing to new biomass via methyl transfer reactions, imparting a large natural 13C-depleted signature. Using 13CO2-labelling, we show that leaf serine, the S-methyl group of leaf methionine, pectin methyl esters, and the associated methanol released during cell wall expansion during growth, are directly produced from photosynthetically-linked C1 metabolism, within minutes of light exposure. We speculate that the photosynthetic C1 pathway is highly conserved across the photosynthetic tree of life, is responsible for synthesis of the greenhouse gas methane, and may have evolved with oxygenic photosynthesis by providing a mechanism of directly linking carbon and ammonia assimilation with growth. Although the rise in atmospheric CO2 inhibits major metabolic pathways like photorespiration, our results suggest that the photosynthetic C1 pathway may accelerate and represents a missing link between enhanced photosynthesis and plant growth rates during CO2 fertilization under a changing climate. A photosynthetic C1 pathway starting with CO2 and NH3 assimilation and ending with methionine synthesis is highly active in foliage of a C3 tree, where it rapidly integrates photosynthesis and C1 metabolism contributing to new biomass via methyl transfer reactions. [ABSTRACT FROM AUTHOR]

Although apparent light inhibition of leaf day respiration is a widespread reported phenomenon, the mechanisms involved, including utilization of alternate respiratory pathways and substrates and light inhibition of TCA cycle enzymes are under active investigation. Recently, acetate fermentation was highlighted as a key drought survival strategy mediated through protein acetylation and jasmonate signaling. Here, we evaluate the light-dependence of acetate transport and assimilation in Populus trichocarpa trees using the dynamic xylem solution injection (DXSI) method developed here for continuous studies of C1 and C2 organic acid transport and light-dependent metabolism. Over 7 days, 1.0 L of [13C]formate and [13C2]acetate solutions were delivered to the stem base of 2-year old potted poplar trees, while continuous diurnal observations were made in the canopy of CO2, H2O, and isoprene gas exchange together with δ13CO2. Stem base injection of 10 mM [13C2]acetate induced an overall pattern of canopy branch headspace 13CO2 enrichment (δ13CO2 +27‰) with a diurnal structure in δ13CO2 reaching a mid-day minimum followed by a maximum shortly after darkening where δ13CO2 values rapidly increased up to +12‰. In contrast, 50 mM injections of [13C]formate were required to reach similar δ13CO2 enrichment levels in the canopy with δ13CO2 following diurnal patterns of transpiration. Illuminated leaves of detached poplar branches pretreated with 10 mM [13C2]acetate showed lower δ13CO2 (+20‰) compared to leaves treated with 10 mM [13C]formate (+320‰), the opposite pattern observed at the whole plant scale. Following dark/light cycles at the leaf-scale, rapid, strong, and reversible enhancements in headspace δ13CO2 by up to +60‰ were observed in [13C2]acetate-treated leaves which showed enhanced dihydrojasmonic acid and TCA cycle intermediate concentrations. The results are consistent with acetate in the transpiration stream as an effective activator of the jasmonate signaling pathway and respiratory substrate. The shorter lifetime of formate relative to acetate in the transpiration stream suggests rapid formate oxidation to CO2 during transport to the canopy. In contrast, acetate is efficiently transported to the canopy where an increased allocation towards mitochondrial dark respiration occurs at night. The results highlight the potential for an effective integration of acetate into glyoxylate and TCA cycles and the light-inhibition of citrate synthase as a potential regulatory mechanism controlling the diurnal allocation of acetate between anabolic and catabolic processes. [ABSTRACT FROM AUTHOR]

A study has been made of the volatile organic compounds (VOCs) released into the contained headspace of packaged lamb's lettuce (Valerianella locusta) plants, cut at the roots, and of separated rocket (Eruca sativa) and baby spinach (Spinacia oleracea) leaves. Direct real‐time analyses of the headspace were carried out on each of five consecutive days using selected‐ion flow‐tube mass spectrometry (SIFT‐MS) and secondary electrospray ionization mass spectrometry (SESI‐MS), together with concomitant off‐line analyses using solid‐phase microextraction (SPME) and gas chromatography mass spectrometry (GC‐MS). It was observed that the concentrations of organosulphur compounds, especially dimethyl sulphide (DMS) and dimethyl disulphide (DMDS) were relatively low in the headspace of fresh samples, but increased exponentially over the 5 days for rocket, increased to a lesser extent for spinach, and increased to an even lesser extent for lamb's lettuce. Methanol was detected in the headspace of even fresh samples of lamb's lettuce, and its concentration increased for the subsequent 2–3 days before decreasing. This trend was mirrored in the isoprenoids analysed, which are characteristic of plant tissues. These results show that direct real‐time VOC analyses by SIFT‐MS can be usefully exploited to monitor fresh leaf salad spoilage, and that SESI‐MS spectra show patterns that are characteristic of salad type and storage time. [ABSTRACT FROM AUTHOR]

The C-urea breath test (C-UBT), developed a few decades ago, is widely used as a non-invasive diagnostic method to detect only the presence of the gastric pathogen Helicobacter pylori infection; however, the actual disease state, i.e. whether the person harbouring H. pylori has peptic ulcer disease (PUD) or non-ulcerous dyspepsia (NUD), is still poorly understood. Nevertheless, the present C-UBT has numerous limitations, drawbacks and pitfalls owing to the ingestion of C-labelled external urea. Here, we show that H. pylori is able to utilize the natural C and O-urea inherently present in the gastric juice in humans for its urease activity which has never been explored before. In vitro measurements of isotopic fractionations of gastric juice urea provide new insights into the actual state of the infection of PUD or NUD. We also provide evidence of the unusual C and O-isotopic fractionations of breath CO that are distinctively altered in individuals with PUD encompassing both gastric and duodenal ulcers as well as with NUD by the enzymatic activity of H. pylori in the gastric niche without oral administration of any C-enriched external urea. This deepens our understanding of the UBT exploiting the natural C and O-gastric juice urea in the pathogenesis of H. pylori infection, reveals the actual disease state of PUD or NUD and thus offers novel opportunities for a simple, robust, cost-effective and non-toxic global strategy devoid of any C-enriched urea for treating these common diseases by a single breath test. [ABSTRACT FROM AUTHOR]

The article discusses a study which investigated the time-dependent excretion kinetics of 18OO/16O isotope ratios of CO2 in exhalted breath samples for the detection of Helicobacter pylori infection in human stomach. Topics discussed include the use of an optical cavity-enhanced integrated cavity output spectroscopy (ICOS) method and the statistical distribution of 18OO/16O.

Hydrogen breath tests (HBT) are widely used for the diagnosis of small intestinal bacterial overgrowth (SIBO) in patients with irritable bowel syndrome (IBS). However, the conclusions drawn from these studies are highly controversial, and several discrepancies exist in the results. The aim of our study is to develop an alternative 13C-glucose breath test (13C-GBT) methodology by measuring high-precision 13CO2/13CO2 isotope ratios in exhaled breath to accurately diagnose SIBO using an optical cavity-enhanced CO2 isotope analyzer. In all, 118 diarrhea-predominant (IBS-D) patients diagnosed according to ROME III criteria underwent 13C-GBT and HBT following the ingestion of a test meal containing 50 mg 13C- enriched glucose with 50 g glucose. The excretion of CO2 enrichments and a cumulative percentage dose of 13C-recovered (c-PDR) significantly depleted (p < 0.001) for the positive SIBO patients (n = 25) compared to the patients with negative SIBO (n = 53) as diagnosed by HBT. A cut-o value of 5.47& at 45 min was indicative of a positive SIBO syndrome. Subsequently, a portion of IBS-D individuals (n=20) whose HBTs were negative but 13C-GBTs were positive, suggesting HBT often fails to diagnose SIBO when the patients may have "non-hydrogen-producing" bacteria. 13C-GBT also correctly diagnosed SIBO in patients (n = 20) within the "grey-zone" and during the preclinical phase of SIBO as opposed to HBT. The prevalence of SIBO with IBS-D in Indian population was estimated to be 45.7%. Our study demonstrates 13C-GBT, for the first time, as a clinically valid and sufficiently robust alternative diagnostic methodology for an accurate evaluation of SIBO in IBS-D patients and superior to HBT. [ABSTRACT FROM AUTHOR]

We report for the first time the excretion kinetics of the percentage dose of C recovered/h (C-PDR %/h) and cumulative PDR, i.e. c-PDR (%) to accomplish the highest diagnostic accuracy of the C-urea breath test (C-UBT) for the detection of Helicobacter pylori infection without any risk of diagnostic errors using an optical cavity-enhanced integrated cavity output spectroscopy (ICOS) method. An optimal diagnostic cut-off point for the presence of H. pylori infection was determined to be c-PDR (%) = 1.47 % at 60 min, using the receiver operating characteristic curve (ROC) analysis to overcome the 'grey zone' containing false-positive and false-negative results of the C-UBT. The present C-UBT exhibited 100 % diagnostic sensitivity (true-positive rate) and 100 % specificity (true-negative rate) with an accuracy of 100 % compared with invasive endoscopy and biopsy tests. Our c-PDR (%) methodology also manifested both diagnostic positive and negative predictive values of 100 %, demonstrating excellent diagnostic accuracy. We also observed that the effect of endogenous CO production related to basal metabolic rates in individuals was statistically insignificant ( p = 0.78) on the diagnostic accuracy. However, the presence of H. pylori infection was indicated by the profound effect of urea hydrolysis rate (UHR). Our findings suggest that the current c-PDR (%) is a valid and sufficiently robust novel approach for an accurate, specific, fast and noninvasive diagnosis of H. pylori infection, which could routinely be used for large-scale screening purposes and diagnostic assessment, i.e. for early detection and follow-up of patients. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The gastric pathogen Helicobacter pylori utilize glucose during metabolism, but the underlying mechanisms linking to oxygen-18 (18O) and carbon-13 (13C)-isotopic fractionations of breath CO2 during glucose metabolism are poorly understood. Using the excretion dynamics of 18O/16O and 13C/12C-isotope ratios of breath CO2, we found that individuals with Helicobacter pylori infections exhibited significantly higher isotopic enrichments of 18O in breath CO2 during the 2h-glucose metabolism regardless of the isotopic nature of the substrate, while no significant enrichments of 18O in breath CO2 were manifested in individuals without the infections. In contrast, the 13C-isotopic enrichments of breath CO2 were significantly higher in individuals with Helicobacter pylori compared to individuals without infections in response to 13C-enriched glucose uptake, whereas a distinguishable change of breath 13C/12C-isotope ratios was also evident when Helicobacter pylori utilize natural glucose. Moreover, monitoring the 18O and 13C-isotopic exchange in breath CO2 successfully diagnosed the eradications of Helicobacter pylori infections following a standard therapy. Our findings suggest that breath 12C18O16O and 13C16O16O can be used as potential molecular biomarkers to distinctively track the pathogenesis of Helicobacter pylori and also for eradication purposes and thus may open new perspectives into the pathogen's physiology along with isotope-specific non-invasive diagnosis of the infection. [ABSTRACT FROM AUTHOR]

The gastric pathogen Helicobacter pylori utilize glucose during metabolism, but the underlying mechanisms linking to oxygen-18 (18O) and carbon-13 (13C)-isotopic fractionations of breath CO2 during glucose metabolism are poorly understood. Using the excretion dynamics of 18O/16O and 13C/12C-isotope ratios of breath CO2, we found that individuals with Helicobacter pylori infections exhibited significantly higher isotopic enrichments of 18O in breath CO2 during the 2h-glucose metabolism regardless of the isotopic nature of the substrate, while no significant enrichments of 18O in breath CO2 were manifested in individuals without the infections. In contrast, the 13C-isotopic enrichments of breath CO2 were significantly higher in individuals with Helicobacter pylori compared to individuals without infections in response to 13C-enriched glucose uptake, whereas a distinguishable change of breath 13C/12C-isotope ratios was also evident when Helicobacter pylori utilize natural glucose. Moreover, monitoring the 18O and 13C-isotopic exchange in breath CO2 successfully diagnosed the eradications of Helicobacter pylori infections following a standard therapy. Our findings suggest that breath 12C18O16O and 13C16O16O can be used as potential molecular biomarkers to distinctively track the pathogenesis of Helicobacter pylori and also for eradication purposes and thus may open new perspectives into the pathogen's physiology along with isotope-specific non-invasive diagnosis of the infection. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase (CA), a well-characterized metalloenzyme, is associated with oxygen-18 ( 18O)-isotopic fractionations of CO2. To investigate how CA activity links the 18O of breath CO2 to pre-diabetes (PD) and type 2 diabetes (T2D) during metabolism, we studied pre- and post-dose CA activities in erythrocytes with simultaneous monitoring of 18O/ 16O-isotope ratios of breath CO2 and thereafter elucidated potential metabolic pathways underlying CA alteration in the pathogenesis of T2D. Here we show that the post-dose CA activity in both T2D and PD was markedly enhanced, whereas the non-diabetic controls (NDC) exhibited a considerable reduction in post-dose CA activity when compared with their basal CA activities. However, T2D and PD exhibited isotopic enrichments of 18O in breath CO2, while a marked depletion of 18O in CO2 was manifested in NDC. Thus, the isotopic enrichments and depletions of 18O in breath CO2 were well correlated with the changes in CA activities for controls, PD and T2D. Our findings suggest the changes in CA activities in erythrocytes may contribute to the pathogenesis of T2D and the breath C 18O 16O regulated by the CA activity as a potential biomarker for non-invasive assessment of T2D, and thus may open a new method for treating T2D. [ABSTRACT FROM AUTHOR]

Traditional leaf gas exchange experiments have focused on net CO 2 exchange (A net ). Here, using California poplar (Populus trichocarpa), we coupled measurements of net oxygen production (NOP), isoprene emissions and δ 18 O in O 2 to traditional CO 2 /H 2 O gas exchange with chlorophyll fluorescence, and measured light, CO 2 and temperature response curves. This allowed us to obtain a comprehensive picture of the photosynthetic redox budget including electron transport rate (ETR) and estimates of the mean assimilatory quotient (AQ = A net /NOP). We found that A net and NOP were linearly correlated across environmental gradients with similar observed AQ values during light (1.25 ± 0.05) and CO 2 responses (1.23 ± 0.07). In contrast, AQ was suppressed during leaf temperature responses in the light (0.87 ± 0.28), potentially due to the acceleration of alternative ETR sinks like lipid synthesis. A net and NOP had an optimum temperature (T opt ) of 31°C, while ETR and δ 18 O in O 2 (35°C) and isoprene emissions (39°C) had distinctly higher T opt . The results confirm a tight connection between water oxidation and ETR and support a view of light-dependent lipid synthesis primarily driven by photosynthetic ATP/NADPH not consumed by the Calvin-Benson cycle, as an important thermotolerance mechanism linked with high rates of (photo)respiration and CO 2 /O 2 recycling., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Stem respiration is a quantitatively important, but poorly understood component of ecosystem carbon cycling in terrestrial ecosystems. However, a dynamic stem gas exchange system for quantifying real-time stem carbon dioxide (CO 2 ) efflux (E s ) is not commercially available resulting in limited observations based on the static method where air is recirculated through a stem enclosure. The static method has limited temporal resolution, suffers from condensation issues, requires a leak-free enclosure, which is often difficult to verify in the field, and requires physically removing the chamber or flushing it with ambient air before starting each measurement.•With the goal of improving our quantitative understanding of biophysical, physiological, biochemical, and environmental factors that influence diurnal E s patterns, here we present a custom system for quantifying real-time stem E s in remote tropical forests.•The system is low cost, lightweight, and waterproof with low power requirements (1.2-2.4 W) for real-time monitoring of stem E s using a 3D printed dynamic stem chamber and a 12V car battery. The design offers control over the flow rate through the stem chamber, eliminates the need for a pump to introduce air into the chamber, and water condensation issues by removing water vapor prior to CO 2 analysis.•Following a simple CO 2 infrared gas analyzer (IRGA) calibration and match procedure with a 400-ppm standard, we quantified diurnal E s observations over a 24-hours period during the summer growing season from an ash tree ( Fraxinus sp.) in Fort Collins, Colorado. The results are consistent with previous laboratory and field studies that show E s can be suppressed during the day relative to the night., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier B.V.)

We report, for the first time, the clinical feasibility of a novel residual gas analyzer mass spectrometry (RGA-MS) method for accurate evaluation of the (13)C-glucose breath test ((13)C-GBT) in the diagnosis of pre-diabetes (PD) and type 2 diabetes mellitus (T2D). In T2D or PD, glucose uptake is impaired and results in blunted isotope enriched (13)CO2 production in exhaled breath samples. Using the Receiver operating characteristics (ROC) curve analysis, an optimal diagnostic cut-off point of the (13)CO2/(12)CO2 isotope ratios expressed as the delta-over-baseline (DOB) value, was determined to be δDOB(13)C‰ = 28.81‰ for screening individuals with non-diabetes controls (NDC) and pre-diabetes (PD), corresponding to a sensitivity of 100% and specificity of 94.4%. We also determined another optimal diagnostic cut-off point of δDOB(13)C‰ = 19.88‰ between individuals with PD and T2D, which exhibited 100% sensitivity and 95.5% specificity. Our RGA-MS methodology for the (13)C-GBT also manifested a typical diagnostic positive and negative predictive value of 96% and 100%, respectively. The diagnostic accuracy, precision and validity of the results were also confirmed by high-resolution optical cavity enhanced integrated cavity output spectroscopy (ICOS) measurements. The δDOB(13)C‰ values measured with RGA-MS method, correlated favourably (R(2) = 0.979) with those determined by the laser based ICOS method. Moreover, we observed that the effects of endogenous CO2 production related to basal metabolic rates in individuals were statistically insignificant (p = 0.37 and 0.73) on the diagnostic accuracy. Our findings suggest that the RGA-MS is a valid and sufficiently robust method for the (13)C-GBT which may serve as an alternative non-invasive point-of-care diagnostic tool for routine clinical practices as well as for large-scale diabetes screening purposes in real-time.