O methoxylamine hydrochloride

Prior to GC-MS analysis, the dry residues were derivatized in two steps. For the methoximation step, the dried aqueous phase samples were suspended in 10μL of 30mg/mL O-methoxylamine hydrochloride dissolved in pyridine (Sigma-Aldrich, St. Louis, MO). After vortexing for 5 minutes, the samples were then incubated on an Eppendorf Thermomixer at 30°C, 1000 rpm for 90 minutes. In the silylation step, 90 µL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) containing 1% trimethylchlorosilane (TMCS, Thermo-Pierce cat. no.TS-48915) was added to each sample. After incubation for 30 minutes at 37°C and 1000 rpm (Eppendorf Thermomixer), the derivatized samples were then transferred into 250μL glass inserts (Agilent) for GC-MS analysis.

Nine individual plants of each line were frozen in liquid N₂ and freeze-dried. Dry plant tissues were ground into fine powders using a tissue-lyzer II (Qiagen, Germany) for 30 s at a frequency of 30 Hz at −80°C. Powdered freeze-dried Arabidopsis leaves or stems (20 mg) were individually suspended in 500 μL of methanol:2-iso-propanol:water (5:2:2, v/v/v). After adding ¹³C labeled ribitol standard (1.5 μg), each material was extracted by vortexing for 10 min, followed by sonication for 5 min. Extracts were individually centrifuged for 10 min at 15,000 × g, with supernatants transferred to a new vial and subsequently dried under vacuum. Dry residues were individually re-suspended in water:acetonitrile (500 μL, 1:1 v/v), and re-extracted by sequential vortexing and sonication, followed by centrifugation at 15,000 × g for 5 min with supernatants then removed and dried under vacuum. Dry residues were individually suspended in O-methoxylamine hydrochloride (40 mg mL^–1 in pyridine, 5 μL, Sigma) and incubated for 90 min at 30°C at 1,000 rpm (Eppendorf Thermomixer). Subsequently, samples were individually derivatized with MSTFA (45 μL) with 1% TMCS (Thermo-Pierce) for 30 min at 37°C and 1,000 rpm (Eppendorf Thermomixer).