Stable Isotope-Assisted Plant Metabolomics: Investigation of Phenylalanine-Related Metabolic Response in Wheat Upon Treatment With the Fusarium Virulence Factor Deoxynivalenol

Abstract

The major Fusarium mycotoxin deoxynivalenol (DON) is a virulence factor in wheat and has also been shown to induce defense responses in host plant tissue. In this study, global and tracer labeling with 13C were combined to annotate the overall metabolome of wheat spikes and to evaluate the response of phenylalanine-related pathways upon treatment with DON. At anthesis, spikes of resistant and susceptible cultivars as well as two related near isogenic wheat lines (NILs) differing in the presence/absence of the major resistance QTL Fhb1 were treated with 1 mg DON or water (control), and samples were collected at 0, 12, 24, 48, and 96 h after treatment (hat). A total of 172 Phe-derived wheat constituents were detected with our untargeted approach employing 13C-labeled phenylalanine and subsequently annotated as flavonoids, lignans, coumarins, benzoic acid derivatives, hydroxycinnamic acid amides (HCAAs), as well as peptides. Ninety-six hours after the DON treatment, up to 30% of the metabolites biosynthesized from Phe showed significantly increased levels compared to the control samples. Major metabolic changes included the formation of precursors of compounds implicated in cell wall reinforcement and presumed antifungal compounds. In addition, also dipeptides, which presumably are products of proteolytic degradation of truncated proteins generated in the presence of the toxin, were significantly more abundant upon DON treatment. An in-depth comparison of the two NILs with correlation clustering of time course profiles revealed some 70 DON-responsive Phe derivatives. While several flavonoids had constitutively different abundance levels between the two NILs differing in resistance, other Phe-derived metabolites such as HCAAs and hydroxycinnamoyl quinates were affected differently in the two NILs after treatment with DON. Our results suggest a strong activation of the general phenylpropanoid pathway and that coumaroyl-CoA is mainly diverted towards HCAAs in the presence of Fhb1, whereas the metabolic route to monolignol(-conjugates), lignans, and lignin seems to be favored in the absence of the Fhb1 resistance quantitative trait loci.

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

“…However, to illustrate the biological relevance, the additionally detected treatment-specific metabolites shall briefly be summarized here. More than one-third of the 62 putative stress-specific compounds (toxin-stressed additional metabolites) are secondary metabolites, most of which were also found in previous studies [38,39], using the same LC-HRMS method. They largely derive from the aromatic amino acids phenylalanine tryptophan and tyrosine [38][39][40].…”

“…More than one-third of the 62 putative stress-specific compounds (toxin-stressed additional metabolites) are secondary metabolites, most of which were also found in previous studies [38,39], using the same LC-HRMS method. They largely derive from the aromatic amino acids phenylalanine tryptophan and tyrosine [38][39][40]. For example, feruloyltryptamine is one of the detected toxin-treatment-specific metabolites, which was identified (identification with confidence level 1 based on the scoring system from [41]).…”

Stable Isotope–Assisted Plant Metabolomics: Combination of Global and Tracer-Based Labeling for Enhanced Untargeted Profiling and Compound Annotation

“…Nevertheless, all detected and annotated metabolites of this manuscript are presented in Supplementary Table 3 . In view of the related, biological study (Doppler et al, 2019), which investigated the Phe-related defense response of wheat against the fungal pathogen F. graminearum , we have selected five respective and representative metabolites to exemplify our isotope-assisted annotation approach.…”

“…The benefits of the additional information obtained from the labeling experiments will be evaluated and discussed in terms of annotation of unknowns, sum formula generation, interpretation of database search and evaluation of MS/MS fragment spectra. In the related article (Doppler et al, 2019) this approach was used to investigate changes in the Phe-submetabolome of wheat upon treatment with the Fusarium graminearum virulence factor and mycotoxin deoxynivalenol.…”

Wheat growth, applied water use efficiency and flag leaf metabolome under continuous and pulsed deficit irrigation

“…Drought stress leads to increased concentrations of BXDs in young wheat seedlings 28 . Moreover, diverse phenylalanine-derived specialised metabolites occur in wheat 29 . Flavonoids and their glycosides are probably particularly relevant under drought stress, as they may help the plants to cope with secondary oxidative stress.…”