Preparation of uniformly labelled 13C- and 15N-plants using customised growth chambers

Ceranic, Asja; Doppler, Maria; Büschl, Christoph; Parich, Alexandra; Xu, Ke; Koutnik, Andrea; Buerstmayr, Hermann; Lemmens, Marc; Schuhmacher, Rainer

doi:10.1186/s13007-020-00590-9

Cited by 17 publications

(12 citation statements)

References 46 publications

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“…The experimental setup used to generate the analyzed data set is shown in Figure 1. Wheat (Triticum durum) has either been grown in a native CO 2 atmosphere (98.9% 12 C) in the glasshouse or in a 13 C-enriched atmosphere (99% 13 C) in a customized cultivation chamber [37] to generate native and uniformly 13 C-labeled (L) wheat, respectively. Both were either treated with water (control and L-control) or toxin (toxin and L-toxin).…”

Section: Resultsmentioning

confidence: 99%

“…To produce globally 13 C-labeled plants, the atmosphere in the labelbox contained 13 CO 2 instead of 12 CO 2 . The complete protocol for wheat cultivation is detailed in [37]. At flowering stage (day 87 after the placement into labelbox), the wheat ears were treated inside the labelbox with the fungal toxin deoxynivalenol (DON) or H 2 O, respectively.…”

Section: Plant Cultivationmentioning

confidence: 99%

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Enhanced Metabolome Coverage and Evaluation of Matrix Effects by the Use of Experimental-Condition-Matched 13C-Labeled Biological Samples in Isotope-Assisted LC-HRMS Metabolomics

et al. 2020

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Stable isotope-assisted approaches can improve untargeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomics studies. Here, we demonstrate at the example of chemically stressed wheat that metabolome-wide internal standardization by globally 13C-labeled metabolite extract (GLMe-IS) of experimental-condition-matched biological samples can help to improve the detection of treatment-relevant metabolites and can aid in the post-acquisition assessment of putative matrix effects in samples obtained upon different treatments. For this, native extracts of toxin- and mock-treated (control) wheat ears were standardized by the addition of uniformly 13C-labeled wheat ear extracts that were cultivated under similar experimental conditions (toxin-treatment and control) and measured with LC-HRMS. The results show that 996 wheat-derived metabolites were detected with the non-condition-matched 13C-labeled metabolite extract, while another 68 were only covered by the experimental-condition-matched GLMe-IS. Additional testing is performed with the assumption that GLMe-IS enables compensation for matrix effects. Although on average no severe matrix differences between both experimental conditions were found, individual metabolites may be affected as is demonstrated by wrong decisions with respect to the classification of significantly altered metabolites. When GLMe-IS was applied to compensate for matrix effects, 272 metabolites showed significantly altered levels between treated and control samples, 42 of which would not have been classified as such without GLMe-IS.

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Section: Resultsmentioning

confidence: 99%

Section: Plant Cultivationmentioning

confidence: 99%

Enhanced Metabolome Coverage and Evaluation of Matrix Effects by the Use of Experimental-Condition-Matched 13C-Labeled Biological Samples in Isotope-Assisted LC-HRMS Metabolomics

et al. 2020

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“…Details for the analysis and data evaluation of the other inspected datasets ( Triticum aestivum , Fusarium graminearum, and Trichoderma reesei ), are given in the Supplementary information (SI 1-4) [10,19,20].…”

Section: Methodsmentioning

confidence: 99%

Heterogeneous multimeric metabolite ion species observed in LC-MS based metabolomics data sets

Abiead¹,

Bueschl

Panzenboeck³

et al. 2022

Preprint

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Covalent or non-covalent heterogeneous multimerization of molecules associated with extracts from biological samples analyzed via LC-MS is quite difficult to recognize/annotate and therefore the prevalence of multimerization remains largely unknown. In this study, we utilized 13C labeled and unlabeled Pichia pastoris extracts to recognize heterogeneous multimers. More specifically, between 0.8% and 1.5% of the biologically-derived features detected in our experiments were confirmed to be heteromers, about half of which we could successfully annotate with monomeric partners. Interestingly, we found specific chemical classes such as nucleotides to disproportionately contribute to heteroadducts. Furthermore, we compiled these compounds into the first MS/MS library that included data from heteromultimers to provide a starting point for other labs to improve the annotation of such ions in other metabolomics data sets. Then, the detected heteromers were also searched in publicly accessible LC-MS datasets available in Metabolights, Metabolomics WB, and GNPS/MassIVE to demonstrate that these newly annotated ions are also relevant to other public datasets. Furthermore, in additional datasets (Triticum aestivum, Fusarium graminearum, and Trichoderma reesei) our developed workflow also detected 0.5% to 4.9% of metabolite features to originate from heterodimers, demonstrating heteroadducts to be present in metabolomics studies at a low percentage.

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“…To this end, the metabolite profiles of the mutants Δrhe2, Δtsc1, and Δtsc2, that lack TOR pathway regulatory proteins, were compared with those of wild type cultures using an isotope-assisted untargeted metabolomics approach based on liquid chromatography-high resolution mass spectrometry (LC-HRMS/MS). Cultivation of the tested strains on native and uniformly 13 Clabeled glucose in the presence of native or ( 15 NH 4 ) 2 SO 4 as a nitrogen source enabled the assignment of both the global and 15 N-containing submetabolomes similar to [47,48]. LC-HRMS measurements and subsequent data evaluation with MetExtract II resulted in a total of 2529 metabolite ions corresponding to 1153 fungal metabolites.…”

Section: Tor Pathway Interference Affects T Atroviride Secondary Metabolite Productionmentioning

confidence: 99%

The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride

et al. 2021

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Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T. atroviride, by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T. atroviride, reduced mycoparasitic overgrowth and, in the case of Δtsc1, a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T. atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T. atroviride.

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Preparation of uniformly labelled 13C- and 15N-plants using customised growth chambers

Cited by 17 publications

References 46 publications

Enhanced Metabolome Coverage and Evaluation of Matrix Effects by the Use of Experimental-Condition-Matched 13C-Labeled Biological Samples in Isotope-Assisted LC-HRMS Metabolomics

Enhanced Metabolome Coverage and Evaluation of Matrix Effects by the Use of Experimental-Condition-Matched 13C-Labeled Biological Samples in Isotope-Assisted LC-HRMS Metabolomics

Heterogeneous multimeric metabolite ion species observed in LC-MS based metabolomics data sets

The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride

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