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Sample preparation<br />
For frozen … Sample preparation<br />
For frozen (F) samples, Arabidopsis plants and pear fruits were frozen in liquid nitrogen immediately after harvesting. They were homogenized using a mixer mill MM310 (Retsch, Haan, Germany) at a frequency of 27 Hz for 3 min at 4°C. Freeze-dried samples, approx. 50 mg of Arabidopsis plants and approx. 100 mg of pear fruits were homogenized to powder using the mixer mill mentioned above. We used three replicates for each sample. Twenty volumes (Arabidopsis) or five volumes (pear) of methanol were added to both F and FD samples (20 mL/mg or 5 mL/mg fresh weight), as well as 8 mM of internal standards (200 mM methionine sulfone for cation analyses, or 200 mM camphor 10-sulfonic acid for anion analyses). The internal standards were used for calibrating the peak area after CE-MS analyses. The samples were homogenized once more at 27 Hz, for 1 min. The sample solutions were then centrifuged at 20 400 x g for 3 min at 4°C. Subsequently, 500 mL chloroform and 200 mL water were added to a 500 mL sample of the supernatant. This mixture was vortexed for 3 min, and centrifuged at 20 400 x g for 3 min at 4°C. The upper layer was evaporated in a centrifugal concentrator for 30 min at 45°C, and then separated into two layers. The upper layer (100–200 mL) was centrifugally filtered through a Millipore 5-kDa cutoff filter at 9100 x g for 150 min. The filtrate was dried in a centrifugal concentrator for 120 min. The residue was dissolved in 20 mL water and used for CE-MS analyses.<br /><br />
Metabolome analysis using CE-MS<br />
Details of the conditions for CE-MS were as described in a previous report. MS were as described in a previous report.
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