Polyphenolic profiling of roots (Vitis spp.) under grape phylloxera (D. vitifoliae Fitch) attack.

Many plants respond to herbivore attacks by the formation of secondary metabolites, such as polyphenols. Grape phylloxera (Daktulosphaira vitifoliaeFitch) induces organoid root galls on fibrous root tips of tolerant Vitis spp. rootstocks. We aim to understand if and how secondary metabolites are involved in the compatible interaction of D. vitifoliae and tolerant Vitis ssp. rootstocks belowground. We hypothise that D. vitifoliae infestation triggers the accumulation of phenolic key compounds in root gall tissue without preventing the compatible host-parasite interaction on two tolerant rootstocks with different genetic background: Teleki 5C (V. berlandieri x V. riparia) and Fercal (B.C. n°1B x 31 Richter). Plants and insects are grown in isolated climate chambers to sample root tips of non-infested plants (control) as well as root tips and galls of D. vitifoliae infested plants (5-14 dai). HPLC-MS-based analyses of phenolic key compounds are compared with gene expression levels of the biosynthetic phenylpropanoid pathway analysing temporal sequences of D. vitifoliae infested root tissue. The results show that the induction of the phenylpropanoid pathway by D. vitifoliae infestation plays an important role in the plant response. Concentrations of phenolic key compounds vary significantly among the rootstocks tested. Both rootstocks display an accumulation of flavan-3-ols and stilbenes in infested root gall tissue. Comparing the host responses of the two rootstocks Fercal shows a stronger accumulation of stilbenes locally in infested root galls, whereas Teleki 5C indicates elevated amounts of stilbenes in non-infested root tip tissue.

MetExtract II: A Software Suite for Stable Isotope-Assisted Untargeted Metabolomics.

Stable isotope labeling (SIL) techniques have the potential to enhance different aspects of liquid chromatography-high-resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics methods including metabolite detection, annotation of unknown metabolites, and comparative quantification. In this work, we present MetExtract II, a software toolbox for detection of biologically derived compounds. It exploits SIL-specific isotope patterns and elution profiles in LC-HRMS(/MS) data. The toolbox consists of three complementary modules: M1 (AllExtract) uses mixtures of uniformly highly isotope-enriched and native biological samples for selective detection of the entire accessible metabolome. M2 (TracExtract) is particularly suited to probe the metabolism of endogenous or exogenous secondary metabolites and facilitates the untargeted screening of tracer derivatives from concurrently metabolized native and uniformly labeled tracer substances. With M3 (FragExtract), tandem mass spectrometry (MS/MS) fragments of corresponding native and uniformly labeled ions are evaluated and automatically assigned with putative sum formulas. Generated results can be graphically illustrated and exported as a comprehensive data matrix that contains all detected pairs of native and labeled metabolite ions that can be used for database queries, metabolome-wide internal standardization, and statistical analysis. The software, associated documentation, and sample data sets are freely available for noncommercial use at http://metabolomics-ifa.boku.ac.at/metextractII .

Metabolism of HT-2 Toxin and T-2 Toxin in Oats.

The Fusarium mycotoxins HT-2 toxin (HT2) and T-2 toxin (T2) are frequent contaminants in oats. These toxins, but also their plant metabolites, may contribute to toxicological effects. This work describes the use of 13C-assisted liquid chromatography-high-resolution mass spectrometry for the first comprehensive study on the biotransformation of HT2 and T2 in oats. Using this approach, 16 HT2 and 17 T2 metabolites were annotated including novel glycosylated and hydroxylated forms of the toxins, hydrolysis products, and conjugates with acetic acid, putative malic acid, malonic acid, and ferulic acid. Further targeted quantitative analysis was performed to study toxin metabolism over time, as well as toxin and conjugate mobility within non-treated plant tissues. As a result, HT2-3-O-ß-d-glucoside was identified as the major detoxification product of both parent toxins, which was rapidly formed (to an extent of 74% in HT2-treated and 48% in T2-treated oats within one day after treatment) and further metabolised. Mobility of the parent toxins appeared to be negligible, while HT2-3-O-ß-d-glucoside was partly transported (up to approximately 4%) through panicle side branches and stem. Our findings demonstrate that the presented combination of untargeted and targeted analysis is well suited for the comprehensive elucidation of mycotoxin metabolism in plants.

MetMatch: A Semi-Automated Software Tool for the Comparison and Alignment of LC-HRMS Data from Different Metabolomics Experiments.

Due to its unsurpassed sensitivity and selectivity, LC-HRMS is one of the major analytical techniques in metabolomics research. However, limited stability of experimental and instrument parameters may cause shifts and drifts of retention time and mass accuracy or the formation of different ion species, thus complicating conclusive interpretation of the raw data, especially when generated in different analytical batches. Here, a novel software tool for the semi-automated alignment of different measurement sequences is presented. The tool is implemented in the Java programming language, it features an intuitive user interface and its main goal is to facilitate the comparison of data obtained from different metabolomics experiments. Based on a feature list (i.e., processed LC-HRMS chromatograms with mass-to-charge ratio (m/z) values and retention times) that serves as a reference, the tool recognizes both m/z and retention time shifts of single or multiple analytical datafiles/batches of interest. MetMatch is also designed to account for differently formed ion species of detected metabolites. Corresponding ions and metabolites are matched and chromatographic peak areas, m/z values and retention times are combined into a single data matrix. The convenient user interface allows for easy manipulation of processing results and graphical illustration of the raw data as well as the automatically matched ions and metabolites. The software tool is exemplified with LC-HRMS data from untargeted metabolomics experiments investigating phenylalanine-derived metabolites in wheat and T-2 toxin/HT-2 toxin detoxification products in barley.

Tracing the metabolism of HT-2 toxin and T-2 toxin in barley by isotope-assisted untargeted screening and quantitative LC-HRMS analysis.

An extensive study of the metabolism of the type A trichothecene mycotoxins HT-2 toxin and T-2 toxin in barley using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) is reported. A recently developed untargeted approach based on stable isotopic labelling, LC-Orbitrap-MS analysis with fast polarity switching and data processing by MetExtract software was combined with targeted LC-Q-TOF-MS(/MS) analysis for metabolite structure elucidation and quantification. In total, 9 HT-2 toxin and 13 T-2 toxin metabolites plus tentative isomers were detected, which were successfully annotated by calculation of elemental formulas and further LC-HRMS/MS measurements as well as partly identified with authentic standards. As a result, glucosylated forms of the toxins, malonylglucosides, and acetyl and feruloyl conjugates were elucidated. Additionally, time courses of metabolite formation and mass balances were established. For absolute quantification of those compounds for which standards were available, the method was validated by determining apparent recovery, signal suppression, or enhancement and extraction recovery. Most importantly, T-2 toxin was rapidly metabolised to HT-2 toxin and for both parent toxins HT-2 toxin-3-O-ß-glucoside was identified (confirmed by authentic standard) as the main metabolite, which reached its maximum already 1 day after toxin treatment. Graphical Abstract Isotope-assisted untargeted screening of HT-2 toxin and T-2 toxin metabolites in barley.

Metabolism of the Fusarium Mycotoxins T-2 Toxin and HT-2 Toxin in Wheat.

To investigate the metabolic fate of HT-2 toxin (HT2) and T-2 toxin (T2) in wheat (Triticum aestivum L.), an untargeted metabolomics study utilizing stable isotopic labeling and liquid chromatography-high resolution mass spectrometry was performed. In total, 11 HT2 and 12 T2 derived in planta biotransformation products were annotated putatively. In addition to previously reported mono- and diglucosylated forms of HT2, evidence for the formation of HT2-malonyl-glucoside and feruloyl-T2, as well as acetylation and deacetylation products in wheat was obtained for the first time. To monitor the kinetics of metabolite formation, a time course experiment was conducted involving the Fusarium head blight susceptible variety Remus and the resistant cultivar CM-82036. Biotransformation reactions were observed already at the earliest tested time point (6 h after treatment), and formed metabolites showed different kinetic profiles. After ripening, less than 15% of the toxins added to the plants were determined to be unmetabolized.