Mulberry fruits are popular in many countries because of their good taste, high nutritional value and medicinal properties. Studying the metabolic profile during fruit development can provide a better understanding of the changes in fruit quality. In this study, a widely targeted metabolomic approach using liquid chromatography‒mass spectrometry (LC‒MS) was used to investigate the widespread metabolic changes during fruit development and ripening in mulberry fruits and combined with multivariate statistical methods to screen for significant changed metabolites (SCMs) at different developmental stages. A total of 981 metabolites in 12 categories were detected in mulberry fruit at three developmental stages (i.e., the fruit expansion stage in which mulberry fruits are green (MG), the fruit color change stage in which they are red (MR) and the fruit ripening stage in which they are purple (MP)). There were significant changes in the metabolic profile at the color change and ripening stages compared to the fruit expansion stage. Flavonoids increased mainly during fruit ripening and accumulated significantly during the mature stage. Large amounts of phenolic acids, lipids, and organic acids accumulated significantly during fruit expansion stage and decreased significantly during ripening. However, sugars accumulate significantly during ripening and are associated with the sweetness of mulberry fruit. Assessment of the levels of different sugar and acid components during fruit development revealed that fructose and glucose were the main sugars, and citric and malic acids were the main organic acids. Meanwhile, the sugar-acid ratio increased significantly, which in turn improved the flavor quality of mulberry fruit. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the SCMs were mainly enriched in galactose metabolism, biosynthesis of secondary metabolites and flavonoid biosynthesis metabolic pathways. The reliability of the metabolomics data was demonstrated by quantitative real-time PCR(qRT‒PCR) validation of six genes encoding key enzymes. The results of this study provide insights into the rational utilization of mulberry fruit resources and the efficient development of functional fruit products.

Aluminum (Al) affects the yield of forest trees in acidic soils. The oil tea plant (Camellia drupifera Lour.) has high Al tolerance, with abundant phenolic compounds in its leaves, especially flavonoid compounds. The role of these flavonoids in the Al resistance of oil tea plants is unclear. In this metabolomic study of C. drupifera under Al stress, ultra-pressure liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was utilized to identify metabolites, while principal component analysis, cluster analysis, and orthogonal partial least squares discriminant analysis were applied to analyze the data on the flavonoid metabolites. The leaf morphology of C. drupifera revealed significant damage by excess aluminum ions under each treatment compared with the control group. Under Al stress at 2 mmol/L (GZ2) and 4 mmol/L (GZ4), the total flavonoid content in C. drupifera leaves reached 24.37 and 35.64 mg/g, respectively, which are significantly higher than the levels measured in the control group (CK) (p < 0.01). In addition, we identified 25 upregulated and 5 downregulated metabolites in the GZ2 vs. CK comparison and 31 upregulated and 7 downregulated flavonoid metabolites in GZ4 vs. CK. The results demonstrate that different levels of Al stress had a significant influence on the metabolite profile of C. drupifera. It was found that the abundance of the 24 differential flavonoid metabolites was gradually elevated with increasing concentrations of Al stress, including catechin, epicatechin, naringenin-7-glucoside, astilbin, taxifolin, miquelianin, quercitrin, and quercimeritrin. Moreover, the most significant increase in antioxidant activity (about 30%) was observed in C. drupifera precultured in leaf extracts containing 7.5 and 15 μg/mL of active flavonoids. The qRT-PCR results showed that the expression levels of key genes involved in the synthesis of flavonoids were consistent with the accumulation trends of flavonoids under different concentrations of Al. Therefore, our results demonstrate the key role of flavonoid compounds in the oil tea plant C. drupifera in response to Al stress, which suggests that flavonoid metabolites in C. drupifera, as well as other aluminum-tolerant plants, may help with detoxifying aluminum.

Mulberry fruits are popular in many countries because of their good taste, high nutritional value and medicinal properties. Studying the metabolic pro le during fruit development can provide a better understanding of the changes in fruit quality. In this study, a widely targeted metabolomic approach using liquid chromatography-mass spectrometry (LC-MS) was used to investigate the widespread metabolic changes during fruit development and ripening in mulberry fruits and combined with multivariate statistical methods to screen for signi cant changed metabolites (SCMs) at different developmental stages. A total of 981 metabolites in 12 categories were detected in mulberry fruit at three developmental stages (i.e., the fruit expansion stage in which mulberry fruits are green (MG), the fruit color change stage in which they are red (MR) and the fruit ripening stage in which they are purple (MP)). There were signi cant changes in the metabolic pro le at the color change and ripening stages compared to the fruit expansion stage. Flavonoids increased mainly during fruit ripening and accumulated signi cantly during the mature stage. Large amounts of phenolic acids, lipids, and organic acids accumulated signi cantly during fruit expansion stage and decreased signi cantly during ripening. However, sugars accumulate signi cantly during ripening and are associated with the sweetness of mulberry fruit. Assessment of the levels of different sugar and acid components during fruit development revealed that fructose and glucose were the main sugars, and citric and malic acids were the main organic acids. Meanwhile, the sugar-acid ratio increased signi cantly, which in turn improved the avor quality of mulberry fruit. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the SCMs were mainly enriched in galactose metabolism, biosynthesis of secondary metabolites and avonoid biosynthesis metabolic pathways. The reliability of the metabolomics data was demonstrated by quantitative real-time PCR(qRT-PCR) validation of six genes encoding key enzymes. The results of this study provide insights into the rational utilization of mulberry fruit resources and the e cient development of functional fruit products.