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.

梨果实发育与成熟过程中的代谢组学数据。
材料与方法
植物材料
供试梨果为西洋梨（Pyrus communis L.‘法兰西’），于2010年和2011年采自日本山形县某私人果园（北纬38°11′，东经140°28′），采集前已获得园主许可。分别于开花前2周（2WBB）、开花前1周（1WBB）、开花当日（B）及开花后2周（2WAB）摘除花托；此外，分别在开花后1个月（1MAB）、2个月（2MAB）、3个月（3MAB）、4个月（4MAB）、收获当日（H，即开花后5个月）以及收获后1个月（1MAH，即成熟果实）采集去皮去籽的梨果，用于后续代谢组学分析。所有样品尽快从果园转运至实验室，随后置于液氮中冷冻保存。将冷冻样品称重后采用匀浆器粉碎，粉碎后的梨果置于离心管中，于-80℃冰箱保存直至提取。每个样品至少设置3次生物学重复。

样品提取
称取10 g粉碎后的冷冻样品，悬浮于50 mL甲醇（MeOH）中，加入内标（internal standard）以校正峰面积，随后以16000×g、4℃条件离心3 min。上清液按分析需求分装：0.5 mL用于离子型代谢物分析，1 mL用于中性代谢物分析，1 mL用于糖类分析，47.5 mL用于植物激素分析。由于早期样品（2WBB、1WBB及B时期）取材量较少，因此将初始样品量调整为1 g，悬浮于20 mL甲醇中提取，后续分装方式同上。提取后的残渣用于淀粉分析。

基于CE-TOF MS的代谢物谱分析
针对离子型代谢物分析，在代谢组学分析前，分别通过氯仿-水液液萃取（liquid–liquid separation）及5 kDa截留超滤管超滤（ultrafiltration），去除疏水化合物与高分子量杂质[6]。采用毛细管电泳-飞行时间质谱（CE-TOF MS）进行离子型代谢物的全面分析，具体方法参照已发表文献[7]。

LC-QTOF-MS非靶向代谢组分析
取含内标的1 mL提取物（阳性内标为2.5 μM利多卡因，阴性内标为10-樟脑磺酸），采用LC-QTOF-MS进行分析（液相系统为Waters Acquity UPLC，质谱系统为Waters Xevo G2 Q-Tof）。分析条件如下：色谱柱为Acquity桥联乙基杂化（BEH）C18柱（1.7 μm，2.1 mm×100 mm，Waters）；流动相体系为流动相A（含0.1%甲酸的水溶液）与流动相B（含0.1%甲酸的乙腈溶液）；梯度洗脱程序为：0 min时99.5%A/0.5%B，0.1 min时99.5%A/0.5%B，10 min时20%A/80%B，10.1 min时0.5%A/99.5%B，12.0 min时0.5%A/99.5%B，12.1 min时99.5%A/0.5%B，15.0 min时99.5%A/0.5%B；流速0.3 mL/min；柱温40℃；质谱检测参数：毛细管电压+3.0 keV，锥孔电压25.0 V，离子源温度120℃，脱溶剂温度450℃，锥孔气流量50 L/h，脱溶剂气流量800 L/h，碰撞能量6 V，质量扫描范围m/z 100~1500，扫描时长0.1 s，扫描间隔0.014 s，数据采集模式为质心模式，正离子模式；Lockspray内标（亮氨酸脑啡肽）扫描时长1.0 s，扫描间隔0.1 s。使用MassLynx V4.1软件（Waters）对数据矩阵进行峰对齐。完成峰对齐、去同位素及低强度峰过滤（强度低于500计数）后，将剩余峰的强度值分别除以利多卡因（[M+H]+，m/z 235.1804）与10-樟脑磺酸（[M-H]-，m/z 231.06910）的强度以完成归一化处理。采用梯度碰撞能量模式采集MS/MS数据，具体参数如下：（1）MS扫描：质量范围m/z 50~1500，扫描时长0.1 s，扫描间隔0.014 s；（2）MS/MS扫描：质量范围m/z 50~1500，扫描时长0.02 s，扫描间隔0.014 s，数据采集模式为质心模式，碰撞能量梯度为10~50 V。在此模式下，自动采集MS扫描中强度前10位（>1000计数）离子的MS/MS谱图；若离子强度低于1000，则不进行MS/MS数据采集。通过解析MS/MS谱图对次生代谢物进行化学鉴定[8]。

糖类与淀粉分析
取用于糖类分析的1 mL甲醇提取物，经真空干燥后溶于0.75 mL去离子水中。向样品溶液中加入等体积的1%（w/v）甘露醇溶液作为内标，混合后用于后续糖类分析。采用高效液相色谱（HPLC）对葡萄糖、果糖、蔗糖和山梨醇进行定量，分析条件如下：色谱柱为Shim-pack SCR101-C柱，柱温70℃；流动相为去离子水，等度洗脱，流速1 mL/min；检测器为示差折光检测器（RI detector，HITACHI L-7490）。针对淀粉分析，使用样品提取后的残渣。参照Murayama等[9]的方法制备醇不溶性残渣。淀粉定量时，先将干燥的醇不溶性残渣悬浮于去离子水中，煮沸30 min。冷却后，使用来自黑曲霉的淀粉葡萄糖苷酶（Roche Applied Science）在50 mM乙酸钠缓冲液（pH 4.5）中酶解凝胶化的淀粉。释放的葡萄糖采用Barham与Trinder的葡萄糖氧化酶-过氧化物酶法进行测定[10]。

基于LC-tripleQ MS的植物激素定量
采用每种激素的稳定同位素内标，对15种植物激素进行检测与定量，具体分析方法参照已发表文献[6]。简要步骤如下：将甲醇提取物真空蒸发后，重悬于5倍体积的含1%乙酸的80%乙腈水溶液中，加入内标与氘代植物激素用于定量，随后置于冰上间歇振荡提取1 h。离心后，沉淀用上述相同体积的溶剂重悬并提取，合并两次上清液。蒸发去除乙腈后，使用固相萃取柱对残留溶液进行纯化。油菜素甾醇类激素需进一步通过HPLC进行纯化。