Labeling Plant Metabolites with Isotopes

Phe and Tyr feeding was performed similarly to the methods of Wang P. et al. (2018 (link)) and Simpson et al. (2021 (link)). Plants were removed from the soil, washed with water, and placed into a 120 ml beaker containing the labeling medium along with two other replicate plants. The labeling medium consisted of 30 ml of a Murashige and Skoog medium supplemented with either 1 mM L-phenylalanine (Sigma) or L-tyrosine, or 1 mM ring-[¹³C₆]-labeled L-phenylalanine or ring-[¹³C₆]-labeled L-tyrosine [Cambridge Isotope Laboratories, Tewskbury, MA. Cat No. CLM-1055 (Phe) or CLM-1542 (Tyr)]. Plants were fed for 24 h in the greenhouse under the conditions described above, after which, each plant was rinsed with water and patted dry. The roots, basal 2 cm of the plant (representing the stem and 2 cm of leaf sheath and developing leaves), and 5 cm of the non-fully expanded leaves were dissected from the plant, flash-frozen in liquid nitrogen, and stored at -70°C until metabolite extraction.