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Tropical peatlands are carbon-dense ecosystems that are significant sources of atmospheric methane (CH4). Recent work has demonstrated the importance of trees as an emission pathway for CH4 from the peat to the atmosphere. However, there remain questions over the processes of CH4 production in these systems and how they relate to substrate supply. Principally, these questions relate to the relative contribution of recent photosynthetically fixed carbon, released as root exudates, versus carbon substrate supply from the slowly decomposing peat matrix to CH4 emissions within these ecosystems. Here, we examined the role of root inputs in regulating CH4 production inferred from soil emissions using a combination of in situ tree girdling, in situ13C natural abundance labelling via stem injections, and a 13CO2 labelling of transplanted plants of two contrasting plant functional types, a broadleaved evergreen tree, and a canopy palm. Girdling of broadleaved evergreen trees reduced CH4 fluxes by up to 67%. Stem injections of trees and palms with a natural abundance label resulted in significant isotopic enrichment of CH4 fluxes, reinforcing the link between root carbon inputs and peat CH4 fluxes. Ex situ13CO2 labelling of plants resulted in significant 13C enrichment of peat CH4 fluxes. Taken together, our results demonstrate for the first time that plant root exudates make a substantial contribution to CH4 production in tropical peatlands. [ABSTRACT FROM AUTHOR]

Identifying metabolites in model organisms is critical for many areas of biology, including unravelling disease aetiology or elucidating functions of putative enzymes. Even now, hundreds of predicted metabolic genes in Saccharomyces cerevisiae remain uncharacterized, indicating that our understanding of metabolism is far from complete even in well-characterized organisms. While untargeted high-resolution mass spectrometry (HRMS) enables the detection of thousands of features per analysis, many of these have a non-biological origin. Stable isotope labelling (SIL) approaches can serve as credentialing strategies to distinguish biologically relevant features from background signals, but implementing these experiments at large scale remains challenging. Here, we developed a SIL-based approach for high-throughput untargeted metabolomics in S. cerevisiae, including deep-48 well format-based cultivation and metabolite extraction, building on the peak annotation and verification engine (PAVE) tool. Aqueous and nonpolar extracts were analysed using HILIC and RP liquid chromatography, respectively, coupled to Orbitrap Q Exactive HF mass spectrometry. Of the approximately 37,000 total detected features, only 3–7% of the features were credentialed and used for data analysis with open-source software such as MS-DIAL, MetFrag, Shinyscreen, SIRIUS CSI:FingerID, and MetaboAnalyst, leading to the successful annotation of 198 metabolites using MS2 database matching. Comparable metabolic profiles were observed for wild-type and sdh1Δ yeast strains grown in deep-48 well plates versus the classical shake flask format, including the expected increase in intracellular succinate concentration in the sdh1Δ strain. The described approach enables high-throughput yeast cultivation and credentialing-based untargeted metabolomics, providing a means to efficiently perform molecular phenotypic screens and help complete metabolic networks. [ABSTRACT FROM AUTHOR]

The major benefits of integrating ion mobility (IM) into LC–MS methods for small molecules are the additional separation dimension and especially the use of IM-derived collision cross sections (CCS) as an additional ion-specific identification parameter. Several large CCS databases are now available, but outliers in experimental interplatform IM-MS comparisons are identified as a critical issue for routine use of CCS databases for identity confirmation. We postulate that different routine external calibration strategies applied for traveling wave (TWIM-MS) in comparison to drift tube (DTIM-MS) and trapped ion mobility (TIM-MS) instruments is a critical factor affecting interplatform comparability. In this study, different external calibration approaches for IM-MS were experimentally evaluated for 87 steroids, for which TWCCSN2, DTCCSN2 and TIMCCSN2 are available. New reference CCSN2 values for commercially available and class-specific calibrant sets were established using DTIM-MS and the benefit of using consolidated reference values on comparability of CCSN2 values assessed. Furthermore, use of a new internal correction strategy based on stable isotope labelled (SIL) internal standards was shown to have potential for reducing systematic error in routine methods. After reducing bias for CCSN2 between different platforms using new reference values (95% of TWCCSN2 values fell within 1.29% of DTCCSN2 and 1.12% of TIMCCSN2 values, respectively), remaining outliers could be confidently classified and further studied using DFT calculations and CCSN2 predictions. Despite large uncertainties for in silico CCSN2 predictions, discrepancies in observed CCSN2 values across different IM-MS platforms as well as non-uniform arrival time distributions could be partly rationalized. [ABSTRACT FROM AUTHOR]

Stable isotope labelling in combination with high-resolution mass spectrometry approaches are increasingly used to analyze both metabolite and protein modification dynamics. To enable correct estimation of the resulting dynamics, it is critical to correct the measured values for naturally occurring stable isotopes, a process commonly called isotopologue correction or deconvolution. While the importance of isotopologue correction is well recognized in metabolomics, it has received far less attention in proteomics approaches. Although several tools exist that enable isotopologue correction of mass spectrometry data, the majority is tailored for the analysis of low molecular weight metabolites. We here present PICor which has been developed for isotopologue correction of complex isotope labelling experiments in proteomics or metabolomics and demonstrate the importance of appropriate correction for accurate determination of protein modifications dynamics, using histone acetylation as an example. [ABSTRACT FROM AUTHOR]

Background and aims: It is unclear if cadmium (Cd) is loaded into cacao beans directly from the roots and stem or rather via reallocation from leaves and stem via the phloem. Here, a split-root experiment with a stable isotope 108Cd tracer was set up to determine the short-term circulation in the vegetative tissues of Cd in Theobroma cacao L. seedlings.Roots of cacao seedlings were split into two parts, each growing in separate containers. The compartments contained an equal volume of nutrient solution with a low-dose Cd concentration of 20 nM, and were either (+) or not (-) enriched in stable 108Cd. Seedlings were grown for two weeks and periodically harvested.On day 1 after labelling, there was a rise in the 108Cd isotopic abundance (IA) in the roots in the labelled compartment whereas the 108Cd IA was unaffected in other plant parts. After 7 days, about 75% of the tracer 108Cd in the plant had been translocated to the shoots and the largest fraction of tracer was recovered in the stem. The 108Cd IA in the roots in the non-labelled compartment rose after day 11, demonstrating that Cd was phloem mobile and reallocated to developing tissues. The isotopic abundance of 108Cd in developing leaves was similar to that in the older leaves, suggesting that Cd originated from leaves rather than directly from roots.We corroborated Cd pathways previously studied in mature cacao trees and demonstrated that Cd is reallocated to sink organs via the phloem.Methods: It is unclear if cadmium (Cd) is loaded into cacao beans directly from the roots and stem or rather via reallocation from leaves and stem via the phloem. Here, a split-root experiment with a stable isotope 108Cd tracer was set up to determine the short-term circulation in the vegetative tissues of Cd in Theobroma cacao L. seedlings.Roots of cacao seedlings were split into two parts, each growing in separate containers. The compartments contained an equal volume of nutrient solution with a low-dose Cd concentration of 20 nM, and were either (+) or not (-) enriched in stable 108Cd. Seedlings were grown for two weeks and periodically harvested.On day 1 after labelling, there was a rise in the 108Cd isotopic abundance (IA) in the roots in the labelled compartment whereas the 108Cd IA was unaffected in other plant parts. After 7 days, about 75% of the tracer 108Cd in the plant had been translocated to the shoots and the largest fraction of tracer was recovered in the stem. The 108Cd IA in the roots in the non-labelled compartment rose after day 11, demonstrating that Cd was phloem mobile and reallocated to developing tissues. The isotopic abundance of 108Cd in developing leaves was similar to that in the older leaves, suggesting that Cd originated from leaves rather than directly from roots.We corroborated Cd pathways previously studied in mature cacao trees and demonstrated that Cd is reallocated to sink organs via the phloem.Results: It is unclear if cadmium (Cd) is loaded into cacao beans directly from the roots and stem or rather via reallocation from leaves and stem via the phloem. Here, a split-root experiment with a stable isotope 108Cd tracer was set up to determine the short-term circulation in the vegetative tissues of Cd in Theobroma cacao L. seedlings.Roots of cacao seedlings were split into two parts, each growing in separate containers. The compartments contained an equal volume of nutrient solution with a low-dose Cd concentration of 20 nM, and were either (+) or not (-) enriched in stable 108Cd. Seedlings were grown for two weeks and periodically harvested.On day 1 after labelling, there was a rise in the 108Cd isotopic abundance (IA) in the roots in the labelled compartment whereas the 108Cd IA was unaffected in other plant parts. After 7 days, about 75% of the tracer 108Cd in the plant had been translocated to the shoots and the largest fraction of tracer was recovered in the stem. The 108Cd IA in the roots in the non-labelled compartment rose after day 11, demonstrating that Cd was phloem mobile and reallocated to developing tissues. The isotopic abundance of 108Cd in developing leaves was similar to that in the older leaves, suggesting that Cd originated from leaves rather than directly from roots.We corroborated Cd pathways previously studied in mature cacao trees and demonstrated that Cd is reallocated to sink organs via the phloem.Conclusions: It is unclear if cadmium (Cd) is loaded into cacao beans directly from the roots and stem or rather via reallocation from leaves and stem via the phloem. Here, a split-root experiment with a stable isotope 108Cd tracer was set up to determine the short-term circulation in the vegetative tissues of Cd in Theobroma cacao L. seedlings.Roots of cacao seedlings were split into two parts, each growing in separate containers. The compartments contained an equal volume of nutrient solution with a low-dose Cd concentration of 20 nM, and were either (+) or not (-) enriched in stable 108Cd. Seedlings were grown for two weeks and periodically harvested.On day 1 after labelling, there was a rise in the 108Cd isotopic abundance (IA) in the roots in the labelled compartment whereas the 108Cd IA was unaffected in other plant parts. After 7 days, about 75% of the tracer 108Cd in the plant had been translocated to the shoots and the largest fraction of tracer was recovered in the stem. The 108Cd IA in the roots in the non-labelled compartment rose after day 11, demonstrating that Cd was phloem mobile and reallocated to developing tissues. The isotopic abundance of 108Cd in developing leaves was similar to that in the older leaves, suggesting that Cd originated from leaves rather than directly from roots.We corroborated Cd pathways previously studied in mature cacao trees and demonstrated that Cd is reallocated to sink organs via the phloem. [ABSTRACT FROM AUTHOR]

Aims: Here we assessed N and P uptake of four grassland species grown together in response to a short-term drought event along a soil P gradient. Methods: We used 15N and 32P tracers to examine uptake of N and P by the grasses Bothriochloa macra, Themeda triandra, Lolium perenne and Microlaena stipoides grown together in pots with initial available P levels of 3, 8, 12, 20 mg P kg−1 soil. Soil moisture in half the pots was reduced from 60 to 30% water holding capacity during a 7-day period to simulate drought. Results: Plant P uptake was strongly reduced by drought in all species across all P levels, much more so than N uptake, indicating decoupling in N and P uptake. Soil available P (Bray method) was not affected by drought, suggesting that the reduced P uptake with drought was due to reduced soil P mobility. Plant competition for N and P changed with drought and soil P levels, where relatively more N and P was taken up with drought by M. stipoides at the lowest soil P level. Conclusions: We showed that greater reductions in P than in N uptake and shifts in N and P uptake among species caused by a short-term drought have strong consequences for plant growth. [ABSTRACT FROM AUTHOR]

Background and aims: One means of reducing nitrate leaching in temperate farming is to include catch crops in crop rotations, which immobilize residual nitrogen (N) in their biomass. For an accurate quantification of the N stored in catch crops and subsequently released from residues, their total biomass, including roots and rhizodeposits has to be assessed. Methods: In a pot experiment under controlled conditions, oil and forage radish ( Raphanus sativus L . var. oleiformis Pers .) and winter turnip rape ( Brassica rapa L . var. silvestris [Lam.] Briggs) plants were leaf-labelled every five to seven days with N-urea (99at%) five times during the vegetation. At harvest, plants were separated into shoot, coarse, medium and fine roots by hand picking and wet sieving, respectively. The amount of N derived from rhizodeposition (NdfR) was calculated using two different calculation approaches. In addition to the pot experiment, a field experiment with unlabelled plants was set up to extrapolate the results from the pot experiment to the field scale, at the same time evaluating the influence of additional mineral N fertilization on biomass distribution. The contribution of rhizodeposition to total N in the field was estimated by extrapolating the root-N-to-rhizodeposition-N ratio of the pot experiment to the field experiment. Results: In the pot trial, between 4.6 and 10.3% of the total assimilated nitrogen of the catch crops was found as rhizodeposits, which is at the lower end of values from other studies on legumes and non-legumes. In the field experiment the shoot-to-root ratio was lower compared to the pot experiment. Thus, the contribution of rhizodeposition to total N under field conditions is substantially higher. Fertilization in the field trial mostly influenced the formation of above-ground plant biomass. Conclusions: Considering the rhizodeposition reveals, that the investigated catch crops store more N than previously assumed. As a consequence, catch crops have to be evaluated with a stronger focus on below-ground biomass to make sure the right amounts of N are considered for fertilization schemes in crop rotations. [ABSTRACT FROM AUTHOR]

Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available C stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for C 'light' and C 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance. [ABSTRACT FROM AUTHOR]

Uptake and cycling of nitrogen (N) in the littoral zone of a lake-receiving nutrient-rich mine water located in Boliden, northern Sweden, was investigated. Stable isotope tracer solutions of N as NH (NAM mesocosm) or NO (NOX mesocosm) were added to mesocosms enclosing plants of common reed ( Phragmites australis). The N abundance in various plant parts was measured at pre-defined time intervals over an experimental period of 22 days. During the course of the experiment, plant parts from the NAM mesocosms were significantly more enriched in N than plant parts from the NOX mesocosms. On day 13, ΔδN values of the fine roots from the NAM mesocosms had reached +8220 ‰, while the maximum ΔδN value in NOX roots was considerably lower at +4430 ‰. Using N values in macrophyte tissues present at the end of the experiment enabled calculations of uptake rates and % of tracer N recovered in the plant (%tracerNrecov). Maximum tracer uptake rates were higher for the NAM mesocosms (1.4 µg g min or 48 mg N m d) compared to the NOX mesocosms (0.23 µg g min or 8.5 mg N m d). Calculations of %tracerNrecov indicated that 1-8 and 25-44 % of added N was assimilated by plants in the NOX and NAM mesocosms, respectively. Hence, P. australis was more effective in assimilating NH, and a larger portion of the tracer N accumulated in the roots compared to the other plant parts. Consequently, macrophyte N removal is most effective for cold-climate aquatic systems receiving mine water dominated by NH. For permanent removal of N, the whole plant (including the roots) should be harvested. [ABSTRACT FROM AUTHOR]

Aims: Natural forests in Ethiopia are frequently replaced by Cupressus lusitanica plantations, but little is known about consequences of this land use change for soil C and N dynamics. The objectives of the study were: (i) quantification of microbial incorporation of litter-derived C and N under field conditions, (ii) identification of forest management effects on microbial incorporation of litter-derived C and N and (iii) elucidation of soil moisture effects on microbial utilization of litter-derived C and N. Methods: Natural litter in the Munessa forest was replaced by C and N labelled litter and its degradation was studied over 2 years. Microbial incorporation of litter-derived C and N was measured by chloroform fumigation extraction and stable isotope analysis. Results: Most of the C and N tracer remained in the litter or was incorporated into bulk soil, whereas soil microbial biomass showed minor incorporation. Silvicultural management practices influenced microbial litter-derived C utilization with increased microbial incorporation under wet soil conditions under plantations. Thinning of Cupressus trees led to increased litter decomposition during dry soil conditions. Conclusion: Soil humidity is the main influencing factor for microbial turnover of litter-derived C in this ecosystem. Fast-growing tree plantations had no negative effects on microbial C and N turnover when compared to natural forests. [ABSTRACT FROM AUTHOR]

Background and Aims: Zn imported into developing cereal grains originates from either de novo Zn uptake by the roots or remobilization of Zn from vegetative tissues. The present study was focused on revealing the quantitative importance of the two pathways for grain Zn loading and how their relative contribution varies with the overall plant Zn status. Methods: The stable isotope Zn was used to trace Zn uptake and remobilization fluxes in barley ( Hordeum vulgare L.) plants growing in hydroponics at 0.1 μM (low Zn), 1.5 μM (medium Zn) or 5 μM Zn (high Zn). When grain development reached 15 days after pollination the Zn source was changed to an enriched Zn isotope and plants were harvested after 6 to 48 h. Zn concentrations and isotope ratios were determined using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Results: Plants with low Zn status absorbed 3-fold more Zn than plants with medium or high Zn status when roots were exposed to an external concentration of 1.5 μM Zn. Stems and ears were the primary recipients of the de novo incorporated Zn with preferential allocation to the developing grains over time. The leaves received in all cases a very small proportion (<5 %) of the newly absorbed Zn and the proportion did not increase over time. Zn fluxes derived from uptake and remobilization were almost equal in plants with low Zn status, while at high Zn status remobilization delivered 4 times more Zn to the developing grains than did root Zn uptake. Conclusions: Stable isotopes in combination with ICP-MS provided a strong tool for quantification of Zn fluxes in intact plants. The importance of Zn remobilization compared to de novo root absorption of Zn increased with increasing plant Zn status. Very little de novo absorbed Zn was translocated to the leaves during generative growth stages. [ABSTRACT FROM AUTHOR]

The protein elicitor from the mycelium of Alternaria tenuissima has been isolated. The elicitor triggered resistance to the tobacco mosaic virus in tobacco by inducing relative oxygen species, but without causing hypersensitive necrosis. The elicitor is reported to impart resistance against Verticillium dahliae and to increase yield in cotton, but its mechanism is not yet clear. In this study, the stable isotope labelled mass spectrometry method was used to quantify the relative abundances of protein expression induced by PeaT1 in Arabidopsis. A significant difference in the relative abundances for the expression of different proteins related to metabolism, modification, regulatory, defense, stress and antioxidation was found in Arabidopsis. [ABSTRACT FROM AUTHOR]

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (13C, 15N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha ( Pichia angusta) as well as a strain of the baker’s yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine’s medium without lactic acid allows relatively low concentrations of 13C and 15N sources, and this medium can be reused several times with supplementation of the 13C source only. [ABSTRACT FROM AUTHOR]

In this minireview the information at the atomic level that has been obtained by studying photosynthetic reaction centers with site-directed isotope labelling is discussed. The required isotopically labelled RCs are prepared using isotopically labelled amino acids and cofactors that have been prepared via organic total synthetic chemistry. In some cases the results of isotopically labelled RCs that are prepared via other methods are also discussed. [ABSTRACT FROM AUTHOR]