Extraction of Polar Metabolite and Measurement by GC-MS

The extraction of polar metabolites was carried out with the protocol described by Hatoum et al. (2014). Briefly, 20 mg of lyophilized mesocarp was placed in a tube containing 500 μl of cold methanol, and 20 μl of 2,910 ng/μl phenyl β-D-glucopyranoside was added as an internal standard. Tubes were then incubated at 70°C for 15 min using a shaking incubator (VorTemp^TM 56, Labnet, Woodbridge, NJ, United States). Tubes were centrifuged for 20 min at 14,000 g and then the precipitate was discarded. One hundred microliters of the supernatant was dried using a stream of nitrogen gas. For derivatization, 120 μl of methoxyamine solution (Sigma-Aldrich, St. Louis, MO, United States) and pyridine 20 mg/ml (Sigma-Aldrich) was added to the dry sample and shaken for 90 min at 30°C. To each tube, 120 μl of BSTFA [N-O-Bis(trimethylsilyl)trifluoroacetamide] (Sigma-Aldrich) was added and shaken for 30 min at 37°C. The content of each tube was transferred into a vial with a micro insert.

Metabolomic analysis was performed by gas chromatography–mass spectrometry (GC-MS). Data were obtained using the protocol described by Fuentealba et al. (2017). Briefly, 1 μl of sample was injected on the GC column of an Agilent GC-MS system (GC7890 with a 5,977 single quadrupole MS with electron impact ionization source; Agilent Technologies, Palo Alto, CA, United States). Each derivatized extract was analyzed twice; a split (1:150) method was used for the abundant compounds such as major sugars and a splitless mode for the less abundant compounds such as organic acids and amino acids. The GC column used was an HP-5-MS capillary column of 30 m length, 0.25 mm internal diameter, and 0.25 μm film thickness (Agilent Technologies). For both methods (split and splitless), the injection and interface temperatures were 220 and 280°C, respectively. Helium was used as carrier gas with a constant flow of 1 ml/min. The GC temperature program started isothermal at 50°C for 1 min (acids method) or at 120°C for 1 min (sugar method) and was then ramped at a rate of 10°C/min to 310°C where it was kept for 13 min (acid method) or to 300°C for 6 min (sugar method). The total run time was 40 min for the acid method and 25 min for the sugar method. Mass spectra in the 50–600 m/z range were recorded at a scanning speed of 2.66 scan cycles per second. The MS ion source and quadrupole temperatures were 230 and 150°C, respectively.

Mass Hunter Data Analysis Software (Agilent Technologies) was used to deconvolute the chromatographic peaks. Identification was performed by comparing the peak retention and mass spectra to the NIST library in the quantitative method. Raw peak area data were corrected using the actual peak area of the internal standard, the sample fresh weight, and a quality control (QC) sample representative of all samples.