Metabolome and Phenylpropanoid Pathway Analysis

Flavonoid molecules detected by LCMS were annotated by their International Chemical Identifier (InChIKey) and separated into subclasses of flavonoids, isoflavonoids, flavones, flavonols, and anthocyanins by ClassyFire (Feunang et al., 2016). For each plant, the average peak height of flavonoids in each plant was calculated and molecules with average peak heights above the mean were considered highly abundant. Significant differences between roots and nodules were identified with two-tailed Welch’s t-tests. A comparison of the overall proportions of flavonoids annotated by class in the metabolomes of D. glomerata and M. sativa was performed with a chi-square test. T-tests and chi-square tests were performed in R using a significance level of p < 0.05.

For molecules with multiple annotated isotopes, only the dominant isotope was used, identified by the highest peak height across all samples. Phenylpropanoid biosynthesis pathways were obtained from KEGG (Kanehisa et al., 2016). Maps used included: Flavonoid Biosynthesis, Flavone and Flavonol Biosynthesis, Anthocyanin Biosynthesis, Isoflavonoid Biosynthesis, and Phenylpropanoid Biosynthesis. Within each class molecules that were significantly different between root and nodule LC-MS samples were grouped by their structural similarity using molecular structures acquired from PubChem (Kim et al., 2016) to reference molecules that included: eriodictyol, naringenin, liquiritigenin, daidzein, genistein, glycitein, formononetin, kaempferol quercetin, luteolin, apigenin, cyanidin, pinocembrin, pinobanksin, galangin, and chrysin. In cases where an enzyme for synthesizing a particular molecule could not be identified, putative pathways were inferred, placing molecules together in groups if their chemical structures shared diagnostic structures of the reference molecules including: a 2C–3C carbon double bond, a 3 carbon hydroxyl, 3′ or 4′ hydroxyls, or a 2 or 3 carbon benzene ring.