Ash leaf metabolomes reveal differences between trees tolerant and susceptible to ash dieback disease

Abstract

European common ash, Fraxinus excelsior, is currently threatened by Ash dieback (ADB) caused by the fungus, Hymenoscyphus fraxineus. To detect and identify metabolites that may be products of pathways important in contributing to resistance against H. fraxineus, we performed untargeted metabolomic profiling on leaves from five high-susceptibility and five low-susceptibility F. excelsior individuals identified during Danish field trials. We describe in this study, two datasets. The first is untargeted LC-MS metabolomics raw data from ash leaves with high-susceptibility and low-susceptibility to ADB in positive and negative mode. These data allow the application of peak picking, alignment, gap-filling and retention-time correlation analyses to be performed in alternative ways. The second, a processed dataset containing abundances of aligned features across all samples enables further mining of the data. Here we illustrate the utility of this dataset which has previously been used to identify putative iridoid glycosides, well known anti-herbivory terpenoid derivatives, and show differential abundance in tolerant and susceptible ash samples.

Figure 1. Experimental workflow from sample processing to feature identification.

(a) Sample processing and mass spectrometry. (b) Data extraction (MassHunter), peak identification, alignment (XCMS) and isotope/adduct annotation (CAMERA). (c) Statistics (JMP and MetaboAnalyst). (d) Fragment ion mass extraction and molecular formula prediction (MassHunter), and feature interpretation(Cytoscape & Molecular Structure Correlator). (e) Feature identification (Various databases and literature searches).

Figure 2. Putative iridoid glycosides as discriminatory features between leaves of F. excelsior genotypes with differential susceptibility to ADB.

(a) Multivariate analysis PLS-DA score plot showing discrimination of five susceptible and five tolerant trees, each replicated three times, based on their leaf metabolite profiles. (b) MS/MS fragmentation network of LC-ESI-MS/MS fragmentation data for discriminatory features of high and low susceptibility genotypes. The size of each circle represents the fragment size. The intensity of blue increases as the number of times each fragment is present in all precursor ions increases. The edges are in shades of red based on retention time; the paler the colour the earlier the retention time. The black (POS) and grey (NEG) features are the discriminatory features. The outside ring shows fragments unique to that precursor ion (i.e., not in the other precursor ions). The inside circle is ‘shared’ fragments, present in more than one precursor ion. Those fragment masses shaded in green have been previously reported from fragmentation of iridoid glycosides. Figure adapted from Sollars et al..

Figure 3. MS-MS Mirror plot of elenolic acid glucoside (ESI-TOF/IT-MS) compared to four negative mode discriminatory features identified in our study (N2, N3, N4 and N5).

The spectra share 4 peaks in common, m/z 179, 223, 371 and 403 (elenolic acid glucoside molecular ion). These fragments correspond to a loss of a methyl and hydroxyl group (403–371), loss of hexose (403–223) which is followed by a loss of CO₂ (223–179). Elenolic acid corresponds to the secoiridoid part of oleuropein-related compounds suggesting that these four features are secoiridoids. Figure from Sollars et al. extended data.

Figure 4. Putative identification of MS-MS fragments for three features observed in negative mode.

Predicted structures for three key m/z peaks using Molecular Structure Correlator (Agilent; http://www.agilent.com/cs/library/usermanuals/public/G3335-90176_MSC_QuickStart.pdf) and the structure of putative IDs. Features of interest were compared to the closest known compounds based on molecular formula and accurate mass. The Excelside A-like feature s demethylated Excelside A. The overall score (top right) and individual fragment scores (below molecular formula) obtained from MSC are shown. Bonds and atoms in black are present in that fragment, whereas grey indicates loss. The molecular formula and the MSC score are displayed next to each fragment structure. CID=collision induced dissociation. Figure adapted from Sollars et al. extended data.