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Sample extraction<br />
Crushed and … Sample extraction<br />
Crushed and frozen samples (10 g) were resuspended in 50 mL of MeOH, including internal standards for standardization of peak areas, and centrifuged (16,000 × g, 3 min, 4°C). Supernatants were dispensed for each analysis as follows: 0.5 mL for ionic metabolites, 1 mL for neutral metabolites, 1 mL for sugars, and 47.5 mL for plant hormones. Because of the small amounts of pear fruit samples obtained in the early periods (2WBB, 1WBB, and B), the initial amounts of these samples were 1 g, extracted in 20 mL of MeOH, and were separately used for each analysis as described above. The residues were subsequently used for starch analysis.<br />
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Sugar and starch analysis<br />
One milliliter of the MeOH extract obtained for sugar analysis was evaporated to dryness and dissolved in 0.75 mL of water. The same volume of 1% (w/v) mannitol (as an internal standard) solution was added to the sample solution, and this mixture was used for subsequent sugar analysis. Sugars (glucose, fructose, sucrose, and sorbitol) were quantified by HPLC according to the conditions described below. The analytical conditions of HPLC were as follows: column, Shim-pack SCR101-C column, 70°C; solvent, distilled water; flow rate, 1 mL/min, isocratic; detection, RI detector (HITACHI L-7490). For starch analysis, the residues of sample extracts were used. An alcohol-insoluble residue was prepared using the method described by Murayama et al. For starch quantification, the dried alcohol-insoluble residue was first suspended in distilled water and boiled for 30 min. After cooling, the gelatinized starch was digested with amyloglucosidase (from Aspergillus niger; Roche Applied Science) in 50 mM sodium acetate buffer (pH 4.5). The released glucose was measured using the glucose oxidase–peroxidase method of Barham and Trinder. e–peroxidase method of Barham and Trinder.
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