Integrative Analyses of Widely Targeted Metabolic Profiling and Transcriptome Data Reveals Molecular Insight into Metabolomic Variations during Apple (Malus domestica) Fruit Development and Ripening

Abstract

The apple is a favorite fruit for human diet and is one of the most important commercial fruit crops around the world. Investigating metabolic variations during fruit development can provide a better understanding on the formation of fruit quality. The present study applied a widely targeted LC-MS-based metabolomics approach with large-scale detection, identification and quantification to investigate the widespread metabolic changes during "Pinova" apple development and ripening. A total of 462 primary and secondary metabolites were simultaneously detected, and their changes along with the four fruit-development stages were further investigated. The results indicated that most of the sugars presented increasing accumulation levels while organic acid, including Tricarboxylic acid cycle (TCA) intermediates, showed a distinct decreasing trend across the four fruit-development stages. A total of 207 secondary metabolites consisted of 104 flavonoids and 103 other secondary metabolites. Many flavonoids maintained relatively high levels in the early fruit stage and then rapidly decreased their levels at the following developmental stages. Further correlation analyses of each metabolite-metabolite pair highlighted the cross talk between the primary and secondary metabolisms across fruit development and ripening, indicating the significant negative correlations between sugars and secondary metabolites. Moreover, transcriptome analysis provided the molecular basis for metabolic variations during fruit development. The results showed that most differentially expressed genes (DEGs) involved in the TCA cycle were upregulated from the early fruit stage to the preripening stage. The extensive downregulation of controlling genes involved in the flavonoid pathway is probably responsible for the rapid decrease of flavonoid content at the early fruit stage. These data provide a global view of the apple metabolome and a comprehensive analysis on metabolomic variations during fruit development, providing a broader and better understanding on the molecular and metabolic basis of important fruit quality traits in commercial apples.

Keywords: Malus domestica; fruit development; fruit quality; metabolome; transcriptome.

Figure 1

Dynamic metabolome of apple development and ripening: (A) Principal component analysis of metabolomics data from four developmental stages of the “Pinova” fruit. “Mix” means the balanced mixture of all fruit samples (quality control). (B) A heat map of the identified metabolites in apple at four developmental stages: The colors indicate the relative content of each identified metabolite among the different developmental stages as determined by the average peak response area by UPLC-MS. (C) Clustering analysis of all 462 metabolites according to their variation tendency during four fruit-development stages and (D) metabolite variation tendencies among the twenty cluster profiles. PS1, 2, 3 and 4 mean the four apple-development stages: PS1 represents 27 Days After Anthesis (DAA); PS2 represents 84 DAA; PS3 represents 125 DAA; and PS4 represents 165 DAA.

Figure 2

Sugar metabolites detected by widely-targeted UPLC-MC during four fruit developmental stages. Four ‘Pinova’ fruit development stages were analyzed: 27 (PS1), 84 (PS2), 125 (PS3) and 165 (PS4) Days After Anthesis (DAA), respectively. The fold changes of each metabolite among these four stages were displayed in heat map which used the first stage of 27 DAA as the calibrator. The Fructose# means that we additionally applied GC-MS to detect fructose content, because we failed to identify fructose by widely-targeted LC-MS metabolomics approach in this study. Three independent replicates were performed for each stage. Significant analysis was performed by SPSS software based on Tukey’s test at p < 0.05 level and showed with lower cases a, b, and c in figure. Different letters indicate significant differences between two samples.

Figure 3

Changes of organic acids and quinate (and its derivatives) during four fruit developmental stages. (A) Heat map of 15 representative organic acids: the fold changes of each metabolite among these four stages were displayed in heat map which used the first stage of 27 Days After Anthesis (DAA) as the calibrator. PS1, 2, 3, and 4 represents four fruit developmental stages: 27, 84, 125, 165 Days After Anthesis (DAA), respectively. Significant analysis was performed by SPSS software based on Tukey’s test at p < 0.05 level and showed with lower cases a, b, and c in figure. Different letters indicate significant differences between two samples. (B) Heat map of organic acids and quinate (and its derivatives): the colors indicate the proportional content of each identified metabolites as determined by the average peak response area with R scale normalization. Three independent replicates were performed for each stage.

Figure 4

Amino acids (left) and their derivates (right) detected by widely-targeted UPLC-MC during four fruit developmental stages. For the amino acids, the fold changes of each metabolite among four stages were displayed in heat map which used the first stage of 27 Days After Anthesis (DAA) as the calibrator. PS1, 2, 3, and 4 represents four fruit developmental stages: 27, 84, 125, 165 Days After Anthesis (DAA), respectively. Significant analysis was performed by SPSS software based on Tukey’s test at p < 0.05 level and showed with lower cases a, b, and c in figure. Different letters indicate significant differences between two samples. For the amino acids derivates, the colors indicate the proportional content of each identified amino acids derivates as determined by the average peak response area with R scale normalization. PS1, 2, 3, and 4 represents four fruit developmental stages: 27, 84, 125, 165 Days After Anthesis (DAA), respectively. Three independent replicates were performed for each stage.

Figure 5

Distributions of accumulation profiles of flavonoids and other secondary metabolites detected by widely targeted UPLC-MC during four fruit-development stages: The colors indicate the proportional content of each identified metabolite as determined by the average peak response area with R scale normalization. PS1, 2, 3 and 4 represent four fruit-development stages: 27, 84, 125 and 165 Days After Anthesis (DAA), respectively. Three independent replicates were performed for each stage.

Figure 6

Representative secondary metabolites detected by widely-targeted UPLC-MC during four fruit developmental stages. The fold changes of each metabolite among these four stages were displayed in heat map which used the first stage of 27 Days After Anthesis (DAA) as the calibrator. PS1, 2, 3, and 4 represents four fruit developmental stages: 27, 84, 125, 165 Days After Anthesis (DAA), respectively. Three independent replicates were performed for each stage. Significant analysis was performed by SPSS software based on Tukey’s test at p < 0.05 level and showed with lower cases a, b, and c in figure. Different letters indicate significant differences between two samples.

Figure 7

A heat map of metabolite–metabolite correlations along with the apple development: 149 representative metabolites were selected from the total 462 metabolites to analyze their correlations. Pearson algorithm was applied to assess metabolite–metabolite correlation coefficients using R software. Each square of the heat map indicates a correlation coefficient score resulting from Pearson analysis, which represents the correlation between the metabolite heading the row and the metabolite heading the column. The 149 representative metabolites for the correlation analysis are listed in Table S2.

Figure 8

Differentially accumulated metabolites among different fruit-development stages: (A) Volcano plots of differential metabolites among PS1 vs. PS2, PS2 vs. PS3 and PS3 vs. PS4; (B) Venn diagram of differential metabolites among PS1 vs. PS2, PS2 vs. PS3 and PS3 vs. PS4; and (C) heat maps of differential metabolites among PS1 vs. PS2, PS2 vs. PS3 and PS3 vs. PS4. Three independent replicates of each stages were also displayed in the heat map. (D) KEGG pathway assignment of differential metabolites among PS1 vs. PS2, PS2 vs. PS3 and PS3 vs. PS4: The dot color represents the p-value, and the dot size represents the number of differential metabolites. PS1, 2, 3 and 4 represent four fruit-development stages: 27, 84, 125 and 165 Days After Anthesis (DAA), respectively.

Figure 9

Metabolomic variations and transcriptional regulations during apple development: The red or blue boxes around each metabolite indicated that the metabolite increased or decreased its levels during fruit development. Each square represented the differentially expressed genes (DEGs) involved in the corresponding pathways based on Mapman bins. The square color indicated the fold change of the DEGs (red means up-regulated and blue is down-regulated). The numbers “1”, “2” and “3” respectively represented the DEGs among PS1 versus (vs.) PS2, PS2 vs. PS3 and PS3 vs. PS4. The red and blue triangles in primary metabolism represented the synthesis and catalyzation of the amino acids, respectively. PS1, 2, 3 and 4 represent four fruit development stages: 27, 84, 125 and 165 Days After Anthesis (DAA), respectively.