Measurement of Photorespiratory Oxygen Exchange by Membrane Inlet Mass Spectrometry.

Photosynthesis and metabolism in plants involve oxygen as both a product and substrate. Oxygen is taken up during photorespiration and respiration and produced through water splitting during photosynthesis. To distinguish between processes that produce or consume O2 in leaves, isotope mass separation and detection by mass spectrometry allows measurement of evolution and uptake of O2 as well as CO2 uptake. This chapter describes how to calculate the rate of Rubisco oxygenation and carboxylation from in vivo gas exchange of stable isotopes of 16O2 and 18O2 with a closed cuvette system for leaf discs and membrane inlet mass spectrometry.

Measurements of Oxygen Evolution in Photosynthesis.

This chapter compares two different techniques for monitoring photosynthetic O2 production; the wide-spread Clark-type O2 electrode and the more sophisticated membrane inlet mass spectrometry (MIMS) technique. We describe how a simple membrane inlet for MIMS can be made out of a commercial Clark-type cell and outline the advantages and drawbacks of the two techniques to guide researchers in deciding which method to use. Protocols and examples are given for measuring O2 evolution rates and for determining the number of chlorophyll molecules per active photosystem II reaction center.

Measurement of O2 Uptake and Evolution in Leaves In Vivo Using Stable Isotopes and Membrane Inlet Mass Spectrometry.

Oxygen is both product and substrate of photosynthesis and metabolism in plants, by oxygen evolution through water splitting and uptake by photorespiration and respiration. It is important to investigate these processes simultaneously in leaves, especially in response to environmental variables, such as light and temperature. To distinguish between processes that evolve or take up O2 in leaves in the light, in vivo gas exchange of stable isotopes of oxygen and membrane inlet mass spectrometry is used. A closed-cuvette system for gas exchange of leaf discs is described, using the stable isotopes 16O2 and 18O2, with a semi-permeable membrane gas inlet and isotope mass separation and detection by mass spectrometry. Measurement of evolution and uptake, as well as CO2 uptake, at a range of light levels allows composition of a light response curve, here described for French bean (Phaseolus vulgaris) and maize (Zea mays) leaf discs.

On the simulation and interpretation of substrate-water exchange experiments in photosynthetic water oxidation.

Water oxidation by photosystem II (PSII) sustains most life on Earth, but the molecular mechanism of this unique process remains controversial. The ongoing identification of the binding sites and modes of the two water-derived substrate oxygens ('substrate waters') in the various intermediates (Si states, i = 0, 1, 2, 3, 4) that the water-splitting tetra-manganese calcium penta-oxygen (Mn4CaO5) cluster attains during the reaction cycle provides central information towards resolving the unique chemistry of biological water oxidation. Mass spectrometric measurements of single- and double-labeled dioxygen species after various incubation times of PSII with H218O provide insight into the substrate binding modes and sites via determination of exchange rates. Such experiments have revealed that the two substrate waters exchange with different rates that vary independently with the Si state and are hence referred to as the fast (Wf) and the slow (WS) substrate waters. New insight for the molecular interpretation of these rates arises from our recent finding that in the S2 state, under special experimental conditions, two different rates of WS exchange are observed that appear to correlate with the high spin and low spin conformations of the Mn4CaO5 cluster. Here, we reexamine and unite various proposed methods for extracting and assigning rate constants from this recent data set. The analysis results in a molecular model for substrate-water binding and exchange that reconciles the expected non-exchangeability of the central oxo bridge O5 when located between two Mn(IV) ions with the experimental and theoretical assignment of O5 as WS in all S states. The analysis also excludes other published proposals for explaining the water exchange kinetics.

Assignment of the slowly exchanging substrate water of nature's water-splitting cofactor.

Identifying the two substrate water sites of nature's water-splitting cofactor (Mn4CaO5 cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S1 state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved 17O-Electron-electron Double resonance detected NMR (TR-17O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H218O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn4CaO5 cofactor after mixing with H217O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from Thermosynechococcus vestitus has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (WS) in the TR-MIMS experiments and ii) that the exchange rates of O5 and WS are both enhanced by Ca2+→Sr2+ substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being WS. This strongly restricts options for the mechanism of water oxidation.

Characterizing compensatory mechanisms in the absence of photoprotective qE in Chlamydomonas reinhardtii.

Rapid fluctuations in the quantity and quality of natural light expose photosynthetic organisms to conditions when the capacity to utilize absorbed quanta is insufficient. These conditions can result in the production of reactive oxygen species and photooxidative damage. Non-photochemical quenching (NPQ) and alternative electron transport are the two most prominent mechanisms which synergistically function to minimize the overreduction of photosystems. In the green alga Chlamydomonas reinhardtii, the stress-related light-harvesting complex (LHCSR) is a required component for the rapid induction and relaxation of NPQ in the light-harvesting antenna. Here, we use simultaneous chlorophyll fluorescence and oxygen exchange measurements to characterize the acclimation of the Chlamydomonas LHCSR-less mutant (npq4lhcsr1) to saturating light conditions. We demonstrate that, in the absence of NPQ, Chlamydomonas does not acclimate to sinusoidal light through increased light-dependent oxygen consumption. We also show that the npq4lhcsr1 mutant has an increased sink capacity downstream of PSI and this energy flow is likely facilitated by cyclic electron transport. Furthermore, we show that the timing of additions of mitochondrial inhibitors has a major influence on plastid/mitochondrial coupling experiments.

Quantitative Detection of Natural Rubber Content in Eucommia ulmoides by Portable Pyrolysis-Membrane Inlet Mass Spectrometry.

Eucommia ulmoides gum (EUG) is a natural polymer predominantly consisting of trans-1,4-polyisoprene. Due to its excellent crystallization efficiency and rubber-plastic duality, EUG finds applications in various fields, including medical equipment, national defense, and civil industry. Here, we devised a portable pyrolysis-membrane inlet mass spectrometry (PY-MIMS) approach to rapidly, accurately, and quantitatively identify rubber content in Eucommia ulmoides (EU). EUG is first introduced into the pyrolyzer and pyrolyzed into tiny molecules, which are then dissolved and diffusively transported via the polydimethylsiloxane (PDMS) membrane before entering the quadrupole mass spectrometer for quantitative analysis. The results indicate that the limit of detection (LOD) for EUG is 1.36 µg/mg, and the recovery rate ranges from 95.04% to 104.96%. Compared to the result of pyrolysis-gas chromatography (PY-GC), the average relative error is 1.153%, and the detection time was reduced to less than 5 min, demonstrating that the procedure was reliable, accurate, and efficient. The method has the potential to be employed to precisely identify the rubber content of natural rubber-producing plants such as Eucommia ulmoides, Taraxacum kok-saghyz (TKS), Guayule, and Thorn lettuce.

Strong variability in nitrogen (N) removal rates in typical agricultural pond from hilly catchment: Evidence from diel and monthly dissolved N2 measurement.

Ponds, depressional submerged landscapes that can store and process nitrogen (N)-enriched runoff from surrounding uplands, are recognized as biogeochemical hotspots for N removal. Despite their strong potential for N removal, information is limited concerning the specifics of their changing nature. Here, we investigated the dynamics of N removal rate in a typical agricultural pond from a hilly catchment, by unraveling the monthly and diel patterns of N2 concentrations and fluxes. Our observations showed that the N pollution in the pond was severe. Its averaged total N level reached 3.6 mg L-1, of which ∼72% consisted of NO3-N. Meanwhile, the water samples were supersaturated with N2, demonstrating N removal occurring in the pond. Further estimates of net N2 fluxes indicated that N removal rates exhibited obvious day-and-night and monthly differences. On the diel scale, N removal rates exhibited a distinct diurnal cycle, with nocturnal rates around 20% higher than during the day. Such a diel pattern can be mainly explained by the fluctuation in DO levels, showing that at nighttime when photosynthesis is absent, low DO environments are conducive to N removal. On a monthly scale, the monthly rates ranged from 0.02 to 0.49 mmol N2 m-2 h-1 (mean: 0.23 mmol N2 m-2 h-1), with generally higher removal rates in the warmer and concurrently rainy months (June-September). N levels in the pond were the corresponding primary explanatory variables. Assembled data from both monthly and hourly scales provided a more complete picture of the changing nature of N removal in ponds. Future work should carefully consider the effects of altered environmental conditions triggered by hydrological events to better reveal the control mechanisms behind the time-immediate N removal from lowland ponds.

Mechanisms of metal-dependent non-redox decarboxylases from quantum chemical calculations.

Quantum chemical calculations are today an extremely valuable tool for studying enzymatic reaction mechanisms. In this mini-review, we summarize our recent work on several metal-dependent decarboxylases, where we used the so-called cluster approach to decipher the details of the reaction mechanisms, including elucidation of the identity of the metal cofactors and the origins of substrate specificity. Decarboxylases are of growing potential for biocatalytic applications, as they can be used in the synthesis of novel compounds of, e.g., pharmaceutical interest. They can also be employed in the reverse direction, providing a strategy to synthesize value-added chemicals by CO2 fixation. A number of non-redox metal-dependent decarboxylases from the amidohydrolase superfamily have been demonstrated to have promiscuous carboxylation activities and have attracted great attention in the recent years. The computational mechanistic studies provide insights that are important for the further modification and utilization of these enzymes in industrial processes. The discussed enzymes are: 5-carboxyvanillate decarboxylase, γ-resorcylate decarboxylase, 2,3-dihydroxybenzoic acid decarboxylase, and iso-orotate decarboxylase.

BeerMIMS: Exploring the Use of Membrane-Inlet Mass Spectrometry (MIMS) Coupled to KNIME for the Characterization of Danish Beers.

Beer is a complex mix of more than 7700 compounds, around 800 of which are volatile. While GC-MS has been actively employed in the analysis of the volatome of beer, this method is challenged by the complex nature of the sample. Herein, we explored the possible of using membrane-inlet mass spectrometry (MIMS) coupled to KNIME to characterize local Danish beers. KNIME stands for Konstanz Information Miner and is a free open-source data processing software which comes with several prebuilt nodes, that, when organized, result in data processing workflows allowing swift analysis of data with outputs that can be visualized in the desired format. KNIME has been shown to be promising in automation of large datasets and requires very little computing power. In fact, most of the computations can be carried out on a regular PC. Herein, we have utilized a KNIME workflow for data visualization of MIMS data to understand the global volatome of beers. Feature identification was not possible as of now but with a combination of MIMS and a KNIME workflow, we were able to distinguish beers from different micro-breweries located in Denmark, laying the foundation for the use of MIMS in future analysis of the beer volatome.

Redirecting photosynthetic electron flux in the cyanobacterium Synechocystis sp. PCC 6803 by the deletion of flavodiiron protein Flv3.

BACKGROUND: Oxygen-evolving photoautotrophic organisms, like cyanobacteria, protect their photosynthetic machinery by a number of regulatory mechanisms, including alternative electron transfer pathways. Despite the importance in modulating the electron flux distribution between the photosystems, alternative electron transfer routes may compete with the solar-driven production of CO2-derived target chemicals in biotechnological systems under development. This work focused on engineered cyanobacterial Synechocystis sp. PCC 6803 strains, to explore possibilities to rescue excited electrons that would normally be lost to molecular oxygen by an alternative acceptor flavodiiron protein Flv1/3-an enzyme that is natively associated with transfer of electrons from PSI to O2, as part of an acclimation strategy towards varying environmental conditions. RESULTS: The effects of Flv1/3 inactivation by flv3 deletion were studied in respect to three alternative end-products, sucrose, polyhydroxybutyrate and glycogen, while the photosynthetic gas fluxes were monitored by Membrane Inlet Mass Spectrometry (MIMS) to acquire information on cellular carbon uptake, and the production and consumption of O2. The results demonstrated that a significant proportion of the excited electrons derived from photosynthetic water cleavage was lost to molecular oxygen via Flv1/3 in cells grown under high CO2, especially under high light intensities. In flv3 deletion strains these electrons could be re-routed to increase the relative metabolic flux towards the monitored target products, but the carbon distribution and the overall efficiency were determined by the light conditions and the genetic composition of the respective pathways. At the same time, the total photosynthetic capacity of the Δflv3 strains was systematically reduced, and accompanied by upregulation of oxidative glycolytic metabolism in respect to controls with the native Flv1/3 background. CONCLUSIONS: The observed metabolic changes and respective production profiles were proposedly linked with the lack of Flv1/3-mediated electron transfer, and the associated decrease in the intracellular ATP/NADPH ratio, which is bound to affect the metabolic carbon partitioning in the flv3-deficient cells. While the deletion of flv3 could offer a strategy for enhancing the photosynthetic production of desired chemicals in cyanobacteria under specified conditions, the engineered target pathways have to be carefully selected to align with the intracellular redox balance of the cells.

In-situ mass spectrometry improves the estimation of stream reaeration from gas-tracer tests.

The estimation of gas-exchange rates between streams and the atmosphere is of great importance for the fate of volatile compounds in rivers. For dissolved oxygen, this exchange process is called reaeration, and its accurate and precise estimation is essential for the quantification of metabolic rates. A common method for the determination of gas-exchange rates is through artificial gas-tracer tests with a proxy gas. We present the implementation of a portable gas-equilibrium membrane inlet mass spectrometer (GE-MIMS) to record concentrations of krypton and propane injected as tracer compound in the context of a gas-tracer test. The field-compatible GE-MIMS uses signals of atmospheric measurements for concentration standardization, and allows recording the dissolved-gas concentrations at a high temporal resolution, leading to overall low measurement uncertainty. Furthermore, the in-situ approach avoids loss of gas during the steps of sampling, transport, storage, and analysis required for ex-situ gas measurements. We compare obtained gas-exchange rate coefficients, reaeration and derived metabolic rates from the in-situ measurements to results obtained from head-space sampling of propane followed by laboratory analysis, and find much lower uncertainties with the in-situ method.

Measurement of O2 Uptake and Evolution in Leaves In Vivo Using Stable Isotopes and Membrane Inlet Mass Spectrometry.

Oxygen is both product and substrate of photosynthesis and metabolism in plants, by oxygen evolution through water splitting and uptake by photorespiration and respiration. It is important to investigate these processes simultaneously in leaves, especially in response to environmental variables, such as light and temperature. To distinguish between processes that evolve or take up O2 in leaves in the light, in vivo gas exchange of stable isotopes of oxygen and membrane inlet mass spectrometry is used. A closed-cuvette system for gas exchange of leaf disks is described, using the stable isotopes 16O2 and 18O2, with a semipermeable membrane gas inlet and isotope mass separation and detection by mass spectrometry. Measurement of evolution and uptake, as well as CO2 uptake, at a range of light levels allows composition of a light-response curve, here described for French bean and maize leaf disks.

Liquid-Phase Measurements of Photosynthetic Oxygen Evolution.

This chapter compares two different techniques for monitoring photosynthetic O2 production: the widespread Clark-type O2 electrode and the more sophisticated membrane inlet mass spectrometry (MIMS) technique. We describe how a simple membrane inlet for MIMS can be made out of a commercial Clark-type cell, and outline the advantages and drawbacks of the two techniques to guide researchers in deciding which method to use. Protocols and examples are given for measuring O2 evolution rates and for determining the number of chlorophyll molecules per active photosystem II reaction center.

Online measurement of phthalate-particulate matter interactions by membrane introduction mass spectrometry (MIMS).

To enable further study and assessment of indoor inhalation exposure risk, an online apparatus enabling measurement of semi-volatile compound partitioning on household particulates was developed. An example for use of the apparatus is described using dimethyl phthalate (DMP). The system employs direct measurement by membrane introduction mass spectrometry (MIMS). The MIMS system was calibrated using known gas phase DMP concentrations produced by gravimetrically calibrated permeation devices. The quantity of DMP sorbed by particles is described first using a model particle type, a reverse-phase liquid chromatography packing material, and then with a household dust sample. In addition, the desorption of semi-volatile compounds from a household dust sample was monitored using the apparatus, and characteristic fragment ion signals for phthalate compounds were observed.

Estimation of Photorespiratory Fluxes by Gas Exchange.

Photorespiratory fluxes can be easily estimated by photosynthetic gas exchange using an infrared gas analyzer and applying the Farquhar, von Caemmerer, and Berry (Farquhar et al. Planta 149:78-90, 1980) photosynthesis model. For a more direct measurement of photorespiratory CO2 release from glycine decarboxylation, infrared gas analysis can be coupled to membrane-inlet mass spectrometry, capable of separating the total CO2 concentration into its 12CO2 and 13CO2 components in a continuous online fashion. This chapter discusses how to calculate rates of photorespiration from Rubisco kinetics and describes in detail a method for measuring the CO2 release from glycine decarboxylation using 13CO2.

Obstacles in the quantification of the cyclic electron flux around Photosystem I in leaves of C3 plants.

Sixty years ago Arnon and co-workers discovered photophosphorylation driven by a cyclic electron flux (CEF) around Photosystem I. Since then understanding the physiological roles and the regulation of CEF has progressed, mainly via genetic approaches. One basic problem remains, however: quantifying CEF in the absence of a net product. Quantification of CEF under physiological conditions is a crucial prerequisite for investigating the physiological roles of CEF. Here we summarize current progress in methods of CEF quantification in leaves and, in some cases, in isolated thylakoids, of C3 plants. Evidently, all present methods have their own shortcomings. We conclude that to quantify CEF in vivo, the best way currently is to measure the electron flux through PS I (ETR1) and that through PS II and PS I in series (ETR2) for the whole leaf tissue under identical conditions. The difference between ETR1 and ETR2 is an upper estimate of CEF, mainly consisting, in C3 plants, of a major PGR5-PGRL1-dependent CEF component and a minor chloroplast NDH-dependent component, where PGR5 stands for Proton Gradient Regulation 5 protein, PGRL1 for PGR5-like photosynthesis phenotype 1, and NDH for Chloroplast NADH dehydrogenase-like complex. These two CEF components can be separated by the use of antimycin A to inhibit the former (major) component. Membrane inlet mass spectrometry utilizing stable oxygen isotopes provides a reliable estimation of ETR2, whilst ETR1 can be estimated from a method based on the photochemical yield of PS I, Y(I). However, some issues for the recommended method remain unresolved.

Acidification, not carbonation, is the major regulator of carbon fluxes in the coccolithophore Emiliania huxleyi.

A combined increase in seawater [CO2 ] and [H(+) ] was recently shown to induce a shift from photosynthetic HCO3 (-) to CO2 uptake in Emiliania huxleyi. This shift occurred within minutes, whereas acclimation to ocean acidification (OA) did not affect the carbon source. To identify the driver of this shift, we exposed low- and high-light acclimated E. huxleyi to a matrix of two levels of dissolved inorganic carbon (1400, 2800 µmol kg(-1) ) and pH (8.15, 7.85) and directly measured cellular O2 , CO2 and HCO3 (-) fluxes under these conditions. Exposure to increased [CO2 ] had little effect on the photosynthetic fluxes, whereas increased [H(+) ] led to a significant decline in HCO3 (-) uptake. Low-light acclimated cells overcompensated for the inhibition of HCO3 (-) uptake by increasing CO2 uptake. High-light acclimated cells, relying on higher proportions of HCO3 (-) uptake, could not increase CO2 uptake and photosynthetic O2 evolution consequently became carbon-limited. These regulations indicate that OA responses in photosynthesis are caused by [H(+) ] rather than by [CO2 ]. The impaired HCO3 (-) uptake also provides a mechanistic explanation for lowered calcification under OA. Moreover, it explains the OA-dependent decrease in photosynthesis observed in high-light grown phytoplankton.

Dissolved atmospheric gas in xylem sap measured with membrane inlet mass spectrometry.

A new method is described for measuring dissolved gas concentrations in small volumes of xylem sap using membrane inlet mass spectrometry. The technique can be used to determine concentrations of atmospheric gases, such as argon, as reported here, or for any dissolved gases and their isotopes for a variety of applications, such as rapid detection of trace gases from groundwater only hours after they were taken up by trees and rooting depth estimation. Atmospheric gas content in xylem sap directly affects the conditions and mechanisms that allow for gas removal from xylem embolisms, because gas can dissolve into saturated or supersaturated sap only under gas pressure that is above atmospheric pressure. The method was tested for red trumpet vine, Distictis buccinatoria (Bignoniaceae), by measuring atmospheric gas concentrations in sap collected at times of minimum and maximum daily temperature and during temperature increase and decline. Mean argon concentration in xylem sap did not differ significantly from saturation levels for the temperature and pressure conditions at any time of collection, but more than 40% of all samples were supersaturated, especially during the warm parts of day. There was no significant diurnal pattern, due to high variability between samples.

Membrane introduction mass spectrometry (MIMS): a versatile tool for direct, real-time chemical measurements.

Membrane introduction mass spectrometry (MIMS) is a direct, continuous, on-line measurement technique. It utilizes a membrane to semi-selectively transfer analyte mixtures from a sample to a mass spectrometer, rejecting the bulk of the sample matrix, which can be a gas, liquid or solid/slurry. Analyte selectivity and sensitivity are affected by optimizations at the membrane, ionization and the mass spectrometer levels. MIMS can be roughly classified by the acceptor phase that entrains analyte(s) to the mass spectrometer after membrane transport, either a gaseous acceptor phase (GP-MIMS) or condensed acceptor phase (CP-MIMS). The aim of this article is to provide an introduction to MIMS as a technique and to explore current variants, recent developments and modern applications, emphasizing examples from our group, the Applied Environmental Research Laboratories as well as selected work from others in this emerging area. Also provided is a synopsis of current and future directions for this versatile analytical technique.