13c6 tyrosine

¹³C₃-lactic acid, ¹³C₂-oxalic acid, ²H₃-sacrosine, ²H₈-valine, ¹³C₃-dihydroxyacetone, ²H₁₀-isoleucine, ¹³C₄-fumaric acid, ¹³C₄-malic acid, ²H₃-aspartic acid, ¹³C₅-glutamic acid, ¹³C₆-4-hydroxybenzoic acid, ²H₃-lauric acid, ¹³C₅-ribose, ¹³C₂-taurine, ²H₄-citric acid, ²H₇-ornithine, ¹³C₆-tyrosine, ¹³C₆-dopa, ²H₆-kynurenine, ²H₈-cystamine, and ¹³C₁₁-tryptophan were purchased from Cambridge Isotope Laboratories, Inc. (MA, USA). ²H₃-2-hydorxybutyric acid and 2-isopropylmalic acid were purchased from CDN isotopes (Quebec, CA) and Sigma Aldrich (Tokyo, Japan), respectively. The compounds were dissolved in methanol, as shown in Supplementary Table 3, and then the obtained solution was used as an extraction solution. A standard alkane series mixture (C7 to C33) and octafluoronaphthalene (OFN) were purchased from Restek Co. (PA, USA) and Shimadzu Co. (Kyoto, Japan), respectively. Methoxyamine hydrochloride and N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), which were used for the derivatization, were obtained from Sigma-Aldrich and GL Science (Tokyo, Japan), respectively.

For feeding experiments, plants were germinated and grown on the Murashige–Skoog (MS) agar medium. Fourteen-day-old seedlings were transferred to MS liquid medium containing 250 μM ¹³C₆-tyrosine (Cambridge Isotope Laboratories). Samples were harvested 24 h later, frozen in liquid nitrogen, and freeze-dried. Ten milligram of freeze-dried samples were extracted with 300 μL of 75% methanol containing 0.5 mg L⁻¹ 4-methylumbelliferone (internal standard) in a sonicator bath for 2 h. The supernatant was hydrolyzed with 1 M HCl at 90°C for 1 h.
pHPL was detected by liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) in negative ionization mode using an Agilent 1260 HPLC and 6460 Triple Quadrupole LC/MS system. An Agilent XDB-C18 column (4.6 × 250 mm, 5 μm particles) was used at 30°C, flow rate of 1 mL/min and with an 18-min linear gradient of 5–70% acetonitrile in 0.1% formic acid. Product ion spectra were used to determine MRM transitions of 187 > 169 for labeled pHPL, 181 > 163 for unlabeled pHPL, and 175 > 133 for 4-methylumbelliferone, respectively.