Although some metabolites can be identified through comparison to authentic standards and interpretation of fragment ions generated by higher-order MS, in untargeted MS, there are still hundreds to thousands of “unknown” metabolite features that have no structural information associated with them. Untargeted MS datasets are rich in information concerning the metabolic repertoires of plants but are insufficient to predict compound structures. The term mass feature rather than compound is appropriate because features can include the parental ions of true metabolites and in-source-generated molecular fragments or adducts. The output of these analyses is a list of mass features, each associated with a mass to charge ratio (m/z), column retention time, and ion count. Untargeted mass spectrometry (MS) can detect and quantify many of the metabolites contained in plant tissues. One potential path for improvement is the formulation of new or modified profiles of metabolites to enhance the resilience of the plant to biotic and abiotic stress. Sorghum is genetically diverse and has a high potential for genetic improvement through breeding ( Brenton et al., 2016). In addition to being a food and forage crop, especially in arid climates, there is interest in improving the properties of sorghum for the biorefinery ( Brenton et al., 2016).

Sorghum bicolor (L.) is a C4 cereal crop known for its adaptation to, and exceptional productivity in, hot and water-limited environments. The Phe- and Tyr-derived metabolite library was also utilized to retrospectively annotate soluble MS features in two brown midrib mutants ( bmr6 and bmr12) identifying several MS features that change significantly in each mutant. In contrast, mass features derived exclusively from Tyr were the most abundant in every tissue. Furthermore, Phe incorporation was greater for many known hydroxycinnamate esters and flavonoid glycosides. In addition, the relative incorporation of Phe and Tyr varied between metabolites and tissues, suggesting the existence of multiple pools of p-coumaric acid that are fed by the two amino acids. Ninety percent of the labeled mass features were derived from both Phe and Tyr, although the proportional contribution of each precursor varied. These features comprised 20% of the MS signal collected by reverse-phase chromatography and detected through negative-ionization. In this study, 13C isotopic-labeled precursors and the recently developed Precursor of Origin Determination in Untargeted Metabolomics (PODIUM) mass spectrometry (MS) analytical pipeline were used to identify over 600 MS features derived from Phe and Tyr in sorghum. Not all derivatives of Phe and Tyr are shared, however, and each can act as a precursor for unique metabolites. Thus, Phe and Tyr are both precursors of common downstream products. Cinnamic acid can, in turn, be converted into p-coumaric acid by cinnamate 4-hydroxylase. Sorghum and other grasses possess phenylalanine ammonia-lyase (PAL) enzymes that generate cinnamic acid from Phe and bifunctional phenylalanine/tyrosine ammonia-lyase (PTAL) enzymes that generate cinnamic acid and p-coumaric acid from Phe and Tyr, respectively.

In addition, the improved characterization of compounds derived from these amino acids could inform strategies for developing crops with greater resilience and improved traits for the biorefinery. Many specialized metabolites are derived from the aromatic amino acids phenylalanine (Phe) and tyrosine (Tyr). The synthesis of small organic molecules, known as specialized or secondary metabolites, is one mechanism by which plants resist and tolerate biotic and abiotic stress.

2Purdue University Center for Plant Biology, West Lafayette, IN, United States.

1Department of Biochemistry, Purdue University, West Lafayette, IN, United States.

Simpson 1 †, Jacob Olson 1 †, Brian Dilkes 1,2 * † and Clint Chapple 1,2 * †

Tyr a space in time