Abstract Korla fragrant pear (Pyrus sinkiangensis Yü) fruits have a unique flavor and are rich in phenolic acids, flavonoids, amino acids, and other nutrients. At present, the molecular basis of the quality differences among Korla fragrant pear fruits with a convex calyx and rough skin (RS), calyx shedding (SD), and a convex calyx (CV) remains unknown. To analyze the main metabolic components of Korla fragrant pear fruits and compare the antioxidant activities of these three fruits with different qualities, we used nutrient composition analysis and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS)-based widely targeted metabolomics approaches to analyze the changes in the quality characteristics of the pulp of these three Korla fragrant pear fruits with different appearances. The nutrient composition analysis showed that the fructose and glucose contents were not significantly different, and sucrose and vitamin C contents were significantly higher in SD fruits compared with CV and RS fruits. However, the levels of flavor substances such as titratable acids, total phenols, and total flavonoids were high in the pulp of RS fruits. The metabolomics results identified 1976 metabolites that were clustered into 12 categories, and phenolic acid and flavonoid metabolites were the most abundant. The differentially accumulated metabolites (DAMs) in the fruits with different appearances were screened by multivariate statistical methods, and a total of 595 DAMs were detected. The analysis identified 300 DAMs between the CV and SD fruits, 246 DAMs between the RS and CV fruits, and 405 DAMs between the RS and SD groups. SD fruits contained the most metabolites with a high relative content, especially phenolic acids, lipids, amino acids and derivatives, alkaloids, and organic acids. Compared with CV fruits, flavonoid metabolism was more active in RS fruits, which also had a higher content of flavonoids, whereas the fewest metabolites were found in CV fruits, which also displayed less flavonoid accumulation. KEGG pathway enrichment analysis revealed that the DAMs were mainly enriched in the metabolic pathways of flavone and flavonol biosynthesis, confirming that CV fruits have decreased flavone and flavonol biosynthesis and accumulate fewer flavonoids than RS fruits, which may explain the less bitter and astringent flavor of CV fruits. However, the flavonoid content in RS fruits was very high, which may be one of the reasons why RS fruits have a harder pulp and are less juicy, more slaggy, and less flavorful. Moreover, the analysis of the antioxidant activity showed that during fruit development and maturation, RS fruits had stronger antioxidant activity than SD and CV fruits. These results provide a theoretical basis for improving the fruit quality of Korla fragrant pears and the processing of pear pulp. Keywords: Korla fragrant pear, UPLC–MS/MS, metabolomics, convex calyx 1. Introduction Korla fragrant pear (Pyrus sinkiangensis Yü) is an endemic economic forest species in China that is mainly distributed in the southern Xinjiang region and has become a high-end, characteristic fruit tree species grown on a large scale in this region [[34]1,[35]2]. Korla fragrant pears fruits are rich in nutrients and contain a variety of mineral elements because of their thin exocarp, crispy flesh, high juice and sugar content, pleasant, rich fragrance, and popularity among consumers [[36]3]. Compared with other Chinese main pear varieties (e.g., Yali pear, Dangshan pear, Jingbai pear, and Nanguo pear), Korla fragrant pear has higher glucose and sucrose contents, and its fructose content is close to that of Nanguo pear [[37]4]. Research has shown that the fragrant pear, compared with other pear varieties, has a relatively low organic acid content that is mainly malic acid. The contents of sugars and organic acids in Korla fragrant pears are significantly higher than those in Yali pear, Dangshan pear, Jingbai pear, and other main planted pear varieties. Therefore, the high sweetness and low organic acid content of Korla fragrant pears are the basis of their high quality and are key characteristics that distinguish them from other pear varieties [[38]4,[39]5]. In P. sinkiangensis Yü, the fruits that are fusiform with a protruding calyx end that is persistent are called calyx convex pears, whereas the fruits that are ovoid with a top that is concave like a funnel and no calyx are called calyx shedding pears. Fruits with a shed calyx have higher contents of soluble sugars and vitamin C and lower contents of titratable acids compared with fruits with a convex calyx [[40]6]. Persistent calyx fruits have the characteristics of an inconsistent fruit shape, more stone cells, large kernels, a small edible portion, a light flavor, and acidity near the kernel, which seriously reduces the economic value of Korla fragrant pears. Among the convex calyx fruits, there are also those that will become rough-skinned with a calyx present. Extensive and in-depth research on fruit calyx shedding and calyx convexity has been carried out with Korla fragrant pears [[41]6,[42]7,[43]8], the affecting factors [[44]9], and the regulatory measures [[45]10,[46]11]. Recently, studies of Korla fragrant pears have focused on calyx abscission, which is significantly affected by auxins. High content of abscisic acid promoted calyx abscission, whereas high content of indole-3-acetic acid and gibberellic acid are detrimental to abscission [[47]12]. The study indicated that spraying paclobutrazol (PP[333]) was effective in increasing calyx abscission rates [[48]13]. Furthermore, the pollination variety also significantly affects the calyx abscission rate of Korla fragrant pear fruits, and research results have confirmed that the calyx abscission rate was higher after pollination with Xueqing, Yali, Zhonglihao, and Cuiguan pears [[49]14]. With the continuous expansion of the Korla fragrant pear cultivation area, the yield has increased substantially; however, fruit quality has declined, and the proportions of convex calyx fruits and rough-skinned fruits have increased. Korla fragrant pear fruits with poor appearance usually have the shortcomings of calyx protrusion, an unattractive appearance, and rough skin. The fruits with convex and rough-skinned calyxes not only have a poor appearance but also hard pulp and a reduced sugar content, often exhibiting characteristics of less juice and more slag, which seriously affect the economic value of Korla fragrant pear and reduce its market competitiveness. However, the molecular basis of the quality differences among pear fruits with convex, shedding, and rough-skinned calyxes remains unknown. Thus, classifying the fruit quality, processing, and utilizing poor-quality fruits are important for improving the industrial value of Korla fragrant pears. Metabolomics is the study of endogenous metabolites in organisms, including organic acids, amino acids, lipids, sugar alcohol compounds, and other substances, analyzing small-molecule metabolites with a molecular mass of less than 1000 [[50]15]. At present, a wide range of targeted metabolomics technologies based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) with excellent chromatographic separation, high sensitivity, and high resolution have been widely used in many fields, such as food science, fruit development, and the evaluation of endogenous plant components [[51]16,[52]17,[53]18,[54]19]. Metabolomics studies on Arabidopsis thaliana and Coreopsis tinctoria from different regions revealed that the growth environment significantly affected the contents and types of amino acids and sugars in A. thaliana and that the flavonoid composition in C. tinctoria grown at high altitude was significantly different from that of C. tinctoria grown at low altitude [[55]19,[56]20]. To reveal the diversity and germplasm-specific flavonoids in radish, the metabolic profiles in the skin and flesh of six colored radish germplasms were analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry. The results revealed that there were significant differences in the flavonoid metabolites among radishes with different qualities, and a total of 133 flavonoids, including 16 dihydroflavones, 44 flavones, 14 flavonoids, nine anthocyanins, and 28 flavonols, were identified [[57]21]. Wang et al. [[58]22] analyzed the metabolites of Lycium barbarum in Ningxia using UPLC–MS/MS technology and found that the types and contents of metabolites of L. barbarum from different origins varied greatly and that external factors, such as temperature and altitude, also altered the types and contents of metabolites in L. barbarum to different degrees. In this study, we used nutrient composition analysis and UPLC-MS/MS-based widely targeted metabolomics approaches to analyze the changes in the quality characteristics of P. sinkiangensis Yü pear fruits with a convex calyx and rough skin (RS), calyx shedding (SD), and a convex calyx (CV). The metabolomic characteristics of the Korla fragrant pear fruits with these three different appearances were assessed by multivariate statistical analysis, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), to understand the mechanisms of pear fruit quality variation. Moreover, the DPPH free radical-scavenging and ABTS cation radical-scavenging capacities were investigated to assess the antioxidant capacity of these fruits with different appearances. The results obtained not only provide information on the differentially accumulated metabolites (DAMs) affecting the quality of Korla fragrant pear fruits but also promote an understanding of how to improve the quality of Korla fragrant pear fruits and the grading, processing, and use of poor-quality fruits. 2. Results 2.1. Morphological Differences among Fruits with a Convex Calyx and Rough Skin (RS), Calyx Shedding (SD), and a Convex Calyx (CV) RS and CV fruits are phenotypically indistinguishable during early fruit development, and changes generally begin to appear later in fruit development. Therefore, RS, SD, and CV fruits were selected at the end of August for metabolomics analysis in this study. As shown in [59]Figure 1, the RS fruits had a persistent calyx with a less beautiful appearance, a more irregular fruit shape, and rougher skin compared with the SD fruits. Figure 1. [60]Figure 1 [61]Open in a new tab Morphologies of fruits with a convex calyx and rough skin, calyx shedding, and a convex calyx. 2.2. Variations in the Main Flavor Substances in Fruits with Different Appearances Fruit flavor is one of the most important factors affecting the organoleptic quality of a product. Therefore, in this study, the contents of sugars (fructose, glucose, and sucrose), titratable acids, vitamin C, and secondary metabolites (total phenols and total flavonoids) were analyzed to identify metabolites associated with changes in fruit flavor ([62]Table S1). The results showed that the fructose and glucose contents of SD fruits were not significantly different compared with CV and RS fruits, but their sucrose contents were significantly higher and increased by 12.98% and 87.54% compared with CV and RS fruits, respectively ([63]Figure 2a–c). The titratable acid content of RS fruits was significantly higher by 76.89% and 27.57% compared with those of SD and CV fruits, respectively ([64]Figure 2d). The vitamin C content in SD fruits was significantly higher than that in CV and RS fruits, whereas the content of secondary metabolites (total phenols and total flavonoids) was significantly higher in RS fruits than in SD and CV fruits ([65]Figure 2e–g). Phenols and flavonoids are important antioxidant substances in different fruits, and a higher content of these metabolites reflects a stronger antioxidant ability [[66]23]. These results indicate that RS fruits have a poorer flavor and decreased quality but better antioxidant activity. Figure 2. [67]Figure 2 [68]Open in a new tab Sugar composition, titratable acids, vitamin C, and major secondary metabolites in the pulp of fruits with different appearances. (a–d) Contents of fructose, glucose, sucrose, and titratable acid in the pulps of the fruits. (e–g) Contents of vitamin C, total phenols, and total flavonoids in the fruit pulps. The error bars represent the means ± SDs (n = 3), and different lowercase letters indicate significant differences (p < 0.05) between fruits with different appearances. 2.3. Metabolic Profiling [69]Figure S1 shows the total ion current (TIC) quality control sample mass spectra obtained by UPLC–MS/MS in the positive and negative ion modes. The spectra show a high degree of overlap, demonstrating that the assay has good signal stability and reliability. The metabolites in the pear fruit pulps from the CV, SD, and RS groups were studied on the basis of UPLC–ESI–MS/MS and databases. A total of 1976 metabolites were detected and identified in the pulps of the fragrant pear fruits with three different appearances. The metabolites were classified into 12 categories, including 359 phenolic acids, 249 others, 244 flavonoids, 242 lipids, 210 amino acids and derivatives, 164 alkaloids, 147 terpenoids, 133 lignans and coumarins, 129 organic acids, 76 nucleotides and derivatives, 16 quinones, and seven tannins ([70]Table S2). Among them, phenolic acids were the most abundant with 18.17% of the total metabolites; others and flavonoids were the next most abundant with 12.60% and 12.35%, respectively; and tannins were the least abundant with 0.35% ([71]Figure 3). These results indicated that the metabolite composition of the pulps from fragrant pear fruits with different appearances varied significantly. Figure 3. [72]Figure 3 [73]Open in a new tab Composition analysis of the identified metabolites. The top five classes of metabolites (phenolic acids, others, flavonoids, lipids, and amino acids and derivatives) are shown next to the chart. The last seven classes are alkaloids, terpenoids, lignans and coumarins, organic acids, nucleotides and derivatives, quinones, tannins. 2.4. Multivariate Statistical Analysis of the Metabolites In this study, the patterns of metabolite accumulation among fruits with different appearances were analyzed by hierarchical clustering, which indicated their differences in expression levels. Most of the phenolic acids, others, flavonoids, and lipids were increased in SD compared with CV and RS. The contents of amino acids and derivatives, alkaloids, terpenoids, lignans, and coumarins were higher in SD than in CV. However, the contents of most organic acids, nucleotides and derivatives, quinones, and tannins were decreased in RS. These data indicated that there was a clear distinction between the SD, CV, and RS fragrant pear fruits ([74]Figure 4a). The overall metabolic differences between the CV, SD, and RS groups and the degree of variability between the samples within each group were discerned by performing PCA on the samples (including the quality control (QC) samples). In [75]Figure 4b, the contributions of PC1 and PC2 were found to be 30.02% and 17.82%, respectively, and the significant difference between each pulp type had a cumulative contribution of 47.84%, which indicated that these two principal components could essentially reflect the main characteristics of the tested samples. In addition, the QC samples were located in the center of the PCA score plot, from which each of the three groups of samples was separated, with tight clustering of the samples within the same group. These data suggested that the results after the data processing of each sample were credible and that there were significant differences between the samples. Similarly, SD differed significantly from the other two fruit appearances, suggesting that the metabolite profile of SD was distinguishable from that of the other two fruits with different appearances and that the metabolite composition in each of the three fruits was highly distinct ([76]Figure S2). Pairwise comparisons of pulp samples were performed using the OPLS-DA model to assess differences between CV and SD (R^2X = 0.652, R^2Y = 0.999, Q^2 = 0.899; [77]Figure 5a), RS and CV (R^2X = 0.523, R^2Y = 1, Q^2 = 0.915; [78]Figure 5b), and RS and SD (R^2X = 0.678, R^2Y = 1, Q^2 = 0.938; [79]Figure 5c). The OPLS-DA score plot showed clear separation of the metabolites from fruits with different appearances, highlighting the great differences between the metabolic profiles of the pulp samples. Moreover, the Q^2 values of all comparison groups were close to 1, indicating that the model is stable and reliable and that differential metabolites can be screened based on VIP values. Figure 4. [80]Figure 4 [81]Open in a new tab Multivariate statistical analysis of the identified metabolites. (a) Clustering heatmap of the identified metabolites. The sample name is listed horizontally, the metabolite information is listed vertically, Group indicates the grouping, and Class indicates the first class of the substance. Different colors are used based on the values obtained after standardization of the relative contents, and the shades reveal the content level, where red represents high content and green represents low content. (b) 2D PCA score plots from the mass spectrometry data of the samples in each group and the quality control samples. Figure 5. [82]Figure 5 [83]Open in a new tab Differential metabolite pairwise comparison OPLS–DA model plots. (a) OPLS–DA model plot for CV vs. SD. (b) OPLS–DA model plot for RS vs. CV. (c) OPLS–DA model plot for RS vs. SD. 2.5. Differential Metabolite Screening and Identification Based on the OPLS-DA results, VIP values > 1, fold change ≥ 2, or fold change ≤ 0.5 were used as criteria to screen the different comparison groups for DAMs ([84]Table S3). A total of 595 DAMs were detected in this study, and the expression levels of these DAMs in the CV and RS groups were significantly different from those in the SD group ([85]Table S4). The results showed that more than half of the DAMs were expressed at high levels in SD and at low levels in CV and RS. The total number of metabolites with a high relative content in SD was the largest, especially phenolic acids, lipids, amino acids and derivatives, alkaloids, and organic acids. Compared with CV, RS had a more active flavonoid metabolism and more flavonoids were present, while the total number of metabolites with a high content was the lowest in CV and the accumulation of flavonoids was lower ([86]Figure 6). The differences in metabolite expression levels in the comparison groups, as well as the statistical significance of the differences, can be clearly observed by volcano plots ([87]Figure 7a–c). There were 300 DAMs between CV and SD (186 downregulated and 114 upregulated), 246 DAMs between RS and CV (128 downregulated and 118 upregulated), and 405 DAMs between RS and SD (264 downregulated and 141 upregulated). Screening of the DAMs showed the greatest differences between SD and the fruits with the other two appearances, followed by RS and CV. These DAMs were classified into 12 different categories, including 114 phenolic acids, 108 flavonoids, 81 lipids, 70 others, 58 amino acids and derivatives, 46 terpenoids, 40 lignans and coumarins, 30 alkaloids, 23 organic acids, 16 nucleotides and derivatives, five quinones, and four tannins. These data show that phenolic acids and flavonoids are the main metabolites. Detailed information on some of the DAMs is listed in [88]Table 1. Furthermore, Venn diagrams were generated from the data from these three pairwise comparisons. As shown in [89]Figure 8, each comparison group had its own specific DAMs. However, there were 35 common DAMs, including 2-hydroxycinnamic acid, chlorogenic acid (3-O-caffeoylquinic acid), naringenin-7-O-rutinoside (narirutin), hesperetin-7-O-rutinoside (hesperidin), LysoPC 12:0, maltotriose, tianshic acid, L-homomethionine, etc. ([90]Table S5). Figure 6. [91]Figure 6 [92]Open in a new tab Clustering heatmap of the DAMs among RS, SD, and CV. The sample name is listed horizontally, the metabolite information is listed vertically, Group indicates the grouping, and Class indicates the first class of the substance. Different colors are used based on the values obtained after standardization of the relative contents, and the shades reveal the content level, where red represents high content and blue represents low content. Tannins are not shown in the figure due to their low number (only 4). Figure 7. [93]Figure 7 [94]Open in a new tab Volcano diagrams of the DAMs. (a) CV vs. SD; (b) RS vs. CV; and (c) RS vs. SD. Each point represents a metabolite, and the horizontal axis represents the log of the fold change of that metabolite between the two groups of samples (log2-fold change). Table 1. Some of the DAMs in CV vs. SD, RS vs. CV, and RS vs. SD. Compound Class Index Compounds Fold Change CV vs. SD RS vs. CV RS vs. SD Phenolic acids Zaln004057 4-caffeoylshikimic acid - 0.38 - mws0178 Chlorogenic acid (3-O-Caffeoylquinic acid) 2.11 0.16 0.33 HJN003 1-O-Sinapoyl-β-D-glucose - 0.37 0.39 mws0853 Sinapyl alcohol 0.44 - - Flavonoids MWSHY0067 Quercetin-3-O-rutinoside (rutin) 0.43 3.52 - MWSHY0046 Quercetin-3-O-glucoside (Isoquercitrin) 0.45 3.94 - Lmjp002596 Quercetin-3-O-sambubioside - 2.14 - mws0913 Kaempferol-3-O-galactoside (Trifolin) - 2.20 - mws0071 Apigenin-4′-O-rhamnoside - 10.30 5.08 Lipids pmp001281 LysoPC 18:1 - - 0.44 pmp001273 LysoPC 18:2 - - 0.43 Lmhp010908 LysoPC 19:1 - - 0.44 Others pme0519 D-Sucrose - 0.50 - Lmsn000381 D-Maltose - 0.49 - Lmxn000398 D-Lactose - 0.49 - mws0232 Riboflavin (Vitamin B2) 0.45 - 0.36 pme0490 Nicotinic acid (Vitamin B3) 0.48 - 0.31 Wasn001007 Isoascorbic acid 2-O-glucoside - - 0.44 MWSmce486 Manninotriose - 2.33 - Zmzn000079 D-erythrose-4-phosphate 2.22 - 2.99 Hmfn000531 L-Ascorbic acid (Vitamin C) 2.20 - 2.65 pma1751 N-(beta-D-Glucosyl)nicotinate 2.38 - 3.36 Amino acids and derivatives Lcsp000959 Gly-Val-Ala - - 0.41 MW0108103 Lys-Phe - - 0.42 mws0250 L-Tyrosine - 0.49 0.41 Terpenoids Wbmn010746 Negundoin A 2.94 - - Cmmn012461 Dehydroabietic acid - 0.30 - Alkaloids mws1375 Nicotianamine 0.45 - - Wbmp003594 6-acetyldelpheline - - 0.46 pmp000727 Feruloylhistamine - - 0.41 Organic acids ZbBn002068 3-methyl-Shikimic acid - - 0.45 Lmbn002072 2-Propylsuccinic acid 0.43 - 0.37 mws0237 Azelaic acid 0.46 - - Lmgn007652 Tianshic acid 10.21 0.23 2.36 mws0376 Fumaric acid - - 2.15 pme3186 DL-Glyceraldehyde-3-phosphate - 2.12 2.22 [95]Open in a new tab Fold change values ≥ 2 or ≤0.5 were considered to indicate significant differences and were used as criteria for screening DAMs. “-” indicates no significant difference. Figure 8. [96]Figure 8 [97]Open in a new tab Venn diagrams of the DAMs in each comparison group from the pulp samples of three pear fruits. 2.6. K-Means Analysis of the DAMs K-means cluster analysis is a commonly used, unsupervised analytical method for grouping samples or metabolites based on their characteristics. To investigate the change trends in the relative contents of metabolites in the fruits with different appearances, the relative contents of all DAMs identified according to the screening criteria in all comparison groups were subjected to unit variance scaling followed by K-means cluster analysis ([98]Figure 9 and [99]Table S6). The annotated DAMs were categorized into eight groups based on their accumulation pattern. Subclass 8 contained 87 metabolites, whose contents in fragrant pear pulp increased as the pulp shape changed, reaching the highest level in RS. This indicates that the DAMs in Subclass 8 are key metabolites in RS pulp. Representative metabolites in this subclass include sinapic acid, 3,4-digalloylshikimic acid, quercetin-7-O-glucoside, naringenin-4′-O-glucoside, dihydrokaempferol-7-O-glucoside, 17-hydroxylinolenic acid, L-asparagine, and L-ascorbic acid (vitamin C), and they were all significantly upregulated in RS vs. SD. Subclasses 2 and 6 consist of 179 metabolites whose contents in the pulp of fragrant pears decreased with changes in the pulp form. Moreover, some DAMs were particularly enriched in the SD (Subclasses 5 and 7), CV (Subclasses 1 and 3), and RS (Subclass 4) groups. Notably, the metabolites in Subclass 8 perfectly reflect the metabolites that are predominantly upregulated. Figure 9. [100]Figure 9 [101]Open in a new tab K-means diagrams of the DAMs. Horizontal coordinates correspond to SD, CV, and RS; vertical coordinates indicate the normalized metabolite relative content; subclass represents the metabolite class number with the same trend. 2.7. KEGG Enrichment Analysis of the DAMs Metabolic pathway enrichment analysis of the screened DAMs was performed with the KEGG platform to understand their change mechanisms in fruits with different appearances. DAMs were annotated with 51 metabolic pathways in CV vs. SD. As shown in [102]Figure 10a, the DAMs were mainly distributed in 20 metabolic pathways, including linoleic acid metabolism, flavone and flavonol biosynthesis, the biosynthesis of secondary metabolites, and sphingolipid metabolism. Among them, two metabolic pathways, linoleic acid metabolism and flavone and flavonol biosynthesis, were significantly enriched (p < 0.05), with six and five DAMs, respectively. In RS vs. CV, the DAMs were annotated to 41 metabolic pathways, such as flavone and flavonol biosynthesis, isoquinoline alkaloid biosynthesis, linoleic acid metabolism, and cutin, suberin, and wax biosynthesis. Among them, only the flavone and flavonol biosynthesis metabolic pathways were significantly enriched (p < 0.05) ([103]Figure 10b). There were five DAMs involved in the flavone and flavonol biosynthesis pathways, including quercetin-3-O-rutinoside (rutin), quercetin-3-O-glucoside (isoquercitrin), quercetin-3-O-sambubioside, kaempferol-3-O-galactoside (trifolin), and kaempferol-3-O-rutinoside (nicotiflorin), and these five DAMs were metabolized more actively and were present in higher contents in RS. The DAMs between RS and SD were annotated to 56 metabolic pathways and were significantly enriched (p < 0.05) in six metabolic pathways, namely, nicotinate and nicotinamide metabolism, tyrosine metabolism, linoleic acid metabolism, phenylpropanoid biosynthesis, stilbenoid, diarylheptanoid, and gingerol biosynthesis, and cyanoamino acid metabolism, where there were eight, eight, eight, eight, two, and four DAMs, respectively ([104]Figure 10c). The results showed that these DAMs were mainly enriched in the metabolic pathways of flavone and flavonol biosynthesis, linoleic acid metabolism, and phenylpropanoid biosynthesis, and that the accumulation of different DAMs in these metabolic pathways might have caused the differences in fruit appearance. Figure 10. [105]Figure 10 [106]Open in a new tab KEGG pathway analysis of the DAMs in CV vs. SD (a), RS vs. CV (b), and RS vs. SD (c). The horizontal axis indicates the corresponding rich factor for each pathway, and the vertical axis gives the name of the pathway (sorted by p value). The color of the dots reflects the size of the p value, with redder indicating more significant enrichment. The size of the dot indicates the number of enriched DAMs. 2.8. Comparison of Antioxidant Activities among Fruits with Different Appearances As shown in [107]Figure 11, the DPPH free radical-scavenging and ABTS cation radical-scavenging capacities of the pulp of Korla fragrant pear fruits showed a decreasing trend during growth and development. RS fruits exhibited the strongest DPPH free radical-scavenging capacity of 29.32 μmol/g at 5 weeks after flower blooming (WAF), which was slightly higher than that of SD and CV fruits. At 15 WAF, RS fruits had a higher DPPH free radical-scavenging capacity than CV fruits. The DPPH free radical-scavenging capacity did not show clear differences among the three fruits at 20 WAF, but the RS fruits had a slightly higher capacity than the SD and CV fruits. The ABTS cation radical-scavenging capacity is an important indicator of the antioxidant effect of a reactive substance. At 5 WAF and 10 WAF, the ABTS cation radical-scavenging capacity of SD fruits was slightly higher than that of RS and CV fruits. At 15 WAF, the ABTS cation radical-scavenging capacity of RS fruits reached 17.75 μmol/g, whereas those of SD and CV fruits reached 12.60 μmol/g and 11.56 μmol/g, respectively. The ABTS cation radical-scavenging capacity at 20 WAF did not show an obvious difference between the three fruits ([108]Table S7). The results showed that the antioxidant activity of RS fruits became stronger during the development and maturity of the fruit, and the extract of this fruit could be used in food processing and other fields. Figure 11. [109]Figure 11 [110]Open in a new tab Comparison of the antioxidant activity among fruits with different appearances at different developmental stages. (a) DPPH-free radical-scavenging capacity. (b) ABTS cation radical-scavenging capacity. 3. Discussion Metabolomics can allow the identification and quantification of all metabolites in a specific tissue or organism that can be considered biomarkers for identifying fruit quality-related factors [[111]24,[112]25]. Among the three metabolomics sequencing technologies, widely targeted metabolomics integrates the advantages of both targeted and nontargeted metabolomics [[113]26]. The UPLC-MS/MS-based approach to widely targeted metabolomics is becoming increasingly popular for analyzing and characterizing plant metabolites as a rapid and reliable methodology [[114]27]. Korla fragrant pear fruits contain a variety of mineral elements and are widely used in jam processing, fruit wine distillation, and functional substance extraction [[115]28]. In addition, fragrant pear fruits have high medicinal value due to their free radical scavenging, anti-inflammatory, analgesic, and antipyretic effects [[116]29]. In Korla fragrant pears, the calyxes of some flowers are deciduous while others are persistent, and persistent calyxes are the main cause of deformed fruits among these pears, which negatively affects their shape and quality and directly affects their economic efficiency [[117]7]. Therefore, we used nutrient composition analysis and UPLC-MS/MS-based widely targeted metabolomics approaches to analyze the changes in the quality characteristics of Korla fragrant pear fruits with a convex calyx and rough skin, calyx shedding, and a convex calyx. Moreover, the DPPH free radical-scavenging and ABTS cation radical-scavenging capacities were used to assess the antioxidant capacity of the fruits with different appearances. The nutrient composition analysis showed that the fructose, glucose, sucrose, and vitamin C contents were significantly higher in SD fruits than in CV and RS fruits. However, high levels of flavor substances such as titratable acids, total phenols, and total flavonoids were found in the pulp of RS fruits. A metabolomics approach was used to measure 1976 metabolites in the pulp of Korla fragrant pears, providing a broader scale with which fragrant pear metabolites could be investigated. These data help to gain insight into the metabolomic landscape of fragrant pears, comprehensively analyze changes in the metabolome during fruit development, and provide a foundation to understand the metabolic basis of the important quality traits in commercial fragrant pears. A total of 595 DAMs were screened by FC and VIP values. These DAMs best represent the differences between the pulp of the Korla fragrant pear fruits with different appearances, which can be used as references for