Metabolic profiling of developing pear fruits reveals dynamic variation in primary and secondary metabolites, including plant hormones

Metabolome data of pear during fruits developing and ripening. Materials and Methods Plant materials Pear fruits (Pyrus communis L. ‘La France’) were harvested from a private orchard in Yamagata Prefecture, Japan (lat. 38°11ʹN and long. 140°28ʹE) in 2010 and 2011 with the permission of the owner. Receptacles were removed at 2 weeks before blooming (2WBB), 1 week before blooming (1WBB), the time of blooming (B), and 2 weeks after blooming (2WAB). Furthermore, peeled and deseeded fruits were collected at 1 month after blooming (1MAB), 2 months after blooming (2MAB), 3 months after blooming (3MAB), 4 months after blooming (4MAB), and the time of harvesting (H; 5 months after blooming) and 1 month after harvesting (1MAH; ripened fruits) and used for subsequent metabolomic analyses. These samples were transferred from the orchard to the laboratory as quickly as possible and were then frozen in liquid nitrogen. The frozen samples were weighed and then crushed using a homogenizer. The crushed pear fruits were placed in conical tubes and stored at –80°C until extraction. At least three biological replicates were prepared for each sample. Sample extraction 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. Metabolite profiling using CE-TOF MS For the analysis of ionic metabolites, hydrophobic and high molecular weight compounds were removed by the preparative processes of liquid­–liquid separation using chloroform and water, and ultrafiltration using a 5 kDa cutoff filter, respectively, prior to the metabolomic analyses [6]. Comprehensive analysis of ionic metabolites using CE-TOF MS was performed as previously reported [7]. Untargeted metabolome analysis by LC-QTOF-MS Extracts (1 mL) containing the internal positive and negative standards 2.5 μM lidocaine and 10-camphorsulfonic acid, respectively, were analyzed using LC-QTOF-MS (LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). Analytical conditions were as follows: LC column, Acquity bridged ethyl hybrid (BEH) C18 (1.7 μm, 2.1 mm × 100 mm, Waters); solvent system, solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid); gradient program, 99.5%A/0.5%B at 0 min, 99.5%A/0.5%B at 0.1 min, 20%A/80%B at 10 min, 0.5%A/99.5%B at 10.1 min, 0.5%A/99.5%B at 12.0 min, 99.5%A/0.5%B at 12.1 min and 99.5%A/0.5%B at 15.0 min; flow rate, 0.3 mL/min; column temperature, 40°C; MS detection: capillary voltage, +3.0 keV, cone voltage, 25.0 V, source temperature, 120°C, desolvation temperature, 450°C, cone gas flow, 50 L/h; desolvation gas flow, 800 L/h; collision energy, 6 V; mass range, m/z 100‒1500; scan duration, 0.1 s; interscan delay, 0.014 s; mode, centroid; polarity, positive; Lockspray (leucine enkephalin): scan duration, 1.0 s; interscan delay, 0.1 s. The data matrix was aligned with MassLynx version 4.1 (Waters). After alignment, deisotoping, and cutoff of low-intensity peaks (fewer than 500 counts), intensity values of the remaining peaks were divided by those of lidocaine ([M+H]+, m/z 235.1804) and 10-camphorsulfonic acid ([M-H]-, m/z 231.06910) for normalization. MS/MS data were acquired in ramp mode under the following analytical conditions: (1) MS: mass range, m/z 50–1500; scan duration, 0.1 s; interscan delay, 0.014 s; and (2) MS/MS: mass range, m/z 50–1500; scan duration, 0.02 s; interscan delay, 0.014 s; data acquisition, centroid mode; collision energy, ramped from 10 to 50 V. In this mode, MS/MS spectra of the top 10 ions (>1000 counts) in an MS scan were automatically obtained. When the ion intensity was less than 1000, MS/MS data acquisition was not performed. The secondary metabolites were chemically assigned by deciphering MS/MS spectra [8]. Sugar and starch analysis 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. [9]. 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 [10]. Quantification of plant hormones using LC-tripleQ MS Fifteen plant hormones were detected and quantified using stable isotopes of each hormone and LC-tripleQ MS analyses were performed as previously reported [6]. Briefly, methanol extracts were evaporated and resuspended in 5 volumes of 80% MeCNaq. containing 1% AcOH with internal standards and deuterated plant hormones, for quantification, and then extracted by mixing occasionally on ice for 1 h. After centrifugation, the pellet was resuspended and extracted with the same volume of the solvent described above, and supernatants were mixed. The MeCN was evaporated and the residual solution was purified using solid-phase extraction columns. Brassinosteroids required further purification by HPLC.