Towards a broader view of the metabolome: untargeted profiling of soluble and bound polyphenols in plants

Phenylalanine (Phe) is a central precursor for numerous secondary plant metabolites with a multitude of biological functions. Recent studies on the fungal disease Fusarium head blight in wheat showed numerous Phe-derived defence metabolites to be induced in the presence of the pathogen. These studies also suggest a partial incorporation of Phe-derived secondary metabolites into the cell wall. To broaden the view of the metabolome to bound Phe derivatives, an existing approach using 13C-labelled Phe as tracer was extended. The developed workflow consists of three successive extractions with an acidified acetonitrile-methanol-water mixture to remove the soluble plant metabolites, followed by cell wall hydrolysis with 4M aqueous NaOH, acidification with aqueous HCl, and liquid-liquid extraction of the hydrolysate with ethyl acetate. The untargeted screening of Phe-derived metabolites revealed 156 soluble compounds and 90 compounds in the hydrolysed samples including known cell wall constituents like ferulic acid, coumaric acid, and tricin. Forty-nine metabolites were found exclusively in the hydrolysate. The average cumulative extraction yield of the soluble metabolites was 99.6%, with a range of 91.8 to 100%. Repeatability coefficients of variation of the protocol ranged from 10.5 to 25.9%, with a median of 16.3%. To demonstrate the suitability of the proposed method for a typical metabolomics application, mock-treated and Fusarium graminearum-treated wheat samples were compared. The study revealed differences between the hydrolysates of the two sample types, confirming the differential incorporation of Phe-derived metabolites into the cell wall under infection conditions.

Automated summary

Phenylalanine is an important precursor for many secondary plant metabolites. Recent studies have shown that defense metabolites derived from phenylalanine are induced in wheat with fungal disease, and some of them are incorporated into the cell wall. A new approach was developed to screen phenylalanine-derived metabolites. The study also demonstrated the suitability of this method for typical metabolomics applications.