Analysis of metabolite differences in skin between Clapp’s Favorite and its mutant Red Clapp’s Favorite through non-targeted metabolomics

Red Clapp’s Favorite is the red mutation cultivar of the pear cultivar Clapp’s Favorite. Fruit color is an important feature of pear fruits, with red skin generally attracting consumers. Anthocyanin, chlorophyll, and carotenoids are the most important pigments in the color formation of fruits. The red color of pear skin is mainly due to the concentration and composition of anthocyanin. Metabolomics is an emerging discipline that focuses on the qualitative and quantitative analysis of small metabolites with low molecular weight in biological cells and tissues. As an important part of systems biology, it is an effective means to solve many complex biological problems. Studies have analyzed pigment content, composition, and differentially expressed genes in the skin of green and red pears from various aspects. Anthocyanins are responsible for physiological activity on regulating pathways. The aim of this study was to discover differential metabolites in the skin of Clapp’s Favorite and its red mutation cultivar Red Clapp’s Favorite. The metabolic components were detected using high-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Chromatographic experiments were performed on an HSS T3 column (100 mm×2.1 mm, 1.8 μm) by using a mobile phase consisting of 0.1% (v/v) formic acid in acetonitrile and water, and mass spectrometry was conducted in the positive and negative modes by electrospray ionization (ESI). Red Clapp’s Favorite and Clapp’s Favorite were collected from the pear germplasm resource nursery of Yantai Institute of Agricultural Sciences in Shandong. The data were analyzed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) as well as cluster analysis and heat map. The first two principal components exhibited 62.3% and 8% of the total variance in the positive and negative ion modes, respectively. PCA can generally reflect metabolite differences between the two groups of samples, and there are significant differences in metabolites between the two cultivars. The results showed that PLS-DA clearly distinguishes the two groups of samples, which can be used to analyze the subsequent difference in components. The compounds were identified based on data retrieved from the PMDB databases according to the accurate mass number, secondary fragment, and isotope distribution. The results showed that the metabolite content in the skin of Red Clapp’s Favorite and Clapp’s Favorite were significant. There were 83 different metabolites (P<0.05, variable importance in project (VIP)≥1), including phenols and amino acids, which are involved in flavonoid metabolism, amino acid metabolism, phenyl propanoid biosynthesis, and other metabolic pathways, including 5 polyphenols, 3 flavonoids, 1 amino acid and derivatives, 8 phenylpropanes, 2 anthocyanins, 5 proanthocyanidins, 6 flavanols, 14 flavonols, 2 isoflavones, 13 triterpenoids, 3 organic acids and derivatives, 1 vitamin, 3 organic acids and derivatives, 15 lipids, and 2 other compounds. The chlorogenic acid and crypto-chlorogenic acid in Red Clapp’s Favorite are 2.40 and 3.46 times as much as those in Clapp’s Favorite. The anthocyanins of cornulin 3-glucoside and cornulin 3-galactoside were 10.235 and 9.394 times, respectively. Phenolic epicatechin and catechin increased by 4.689 and 4.635, respectively. The content of phenylpropane 3, 4-dihydroxycinnamic acid in Red Clapp’s Favorite increased by 3.13 times. Among the 83 differential metabolites, 23 metabolites were enriched in the pathway. To display the relationship between the samples and the differences in metabolites among the different samples intuitively, hierarchical clustering and heat map analysis were performed on the metabolite expression levels with significant differences in the enrichment pathways. The Kyoto Encyclopedia of Genes and Genomes database was used to further analyze the pathway enrichment of different metabolites. According to the results, there were 6 metabolic pathways (P<0.05): flavonoid biosynthesis, flavone and flavonol biosynthesis, phenylpropanoid biosynthesis, butanoate metabolism, phenylalanine metabolism, and tyrosine metabolism. Plant secondary metabolism shows a complex diversity. This study would screen out other pathways affecting the biosynthesis of flavonoids, which could provide reference for the further study of biosynthesis and biological function of flavonoids in red fruits. This study provides a useful reference for metabolomics of red pears, which could provide a theoretical reference for the quality analysis and biological function research of pears.