Integrated metabolomics and transcriptomics unravel the biosynthZaesis mechanism of anthocyanin in postharvest red raspberry (Rubus idaeus L.)

Abstract

Introduction: Anthocyanins are crucial secondary metabolites that are responsible for pigment deposition in fruits. Raspberry fruit color shifts from white to red during natural or postharvest ripening. However, the precise mechanisms and biosynthetic pathways of anthocyanins in postharvest raspberries remain unclear.

Methods: This study used metabolomic and transcriptomic analyses to explore anthocyanin biosynthesis in postharvest raspberries at various color stages: white (RBT-1), white-to-pink (RBT-2), pink (RBT-3), red (RBT-4), and deep red (RBT-5).

Results: We identified 43 key metabolites, and 13,239 DEGs linked to anthocyanin biosynthesis in postharvest raspberry colour development, including cyanidin-3-O-sophoroside and cyanidin-3-O-glucoside. The key DAMs in colored raspberries were gentiobioside, pelargonidin-3,5-O-diglucoside, cyanidin-3-O-sambubioside, and pelargonidin-3-O-sambubioside. Transcriptome analysis revealed 32 differentially expressed structural genes linked to anthocyanin and flavonoid synthesis, with significant upregulation of PAL, CHS, F3H, C4H, F3'H, DFR, ANS, CHI, and UFGT genes, which promote anthocyanin synthesis and pigment accumulation. Integrated analysis showed that cyanidin-3-O-sophoroside was correlated with 9 structural genes involved in anthocyanin biosynthesis, 19 transcription factors (TFs), and 14 hormone signaling-related genes.

Discussion: This study explored the regulatory mechanisms of MYB, WRKY, bHLH, and NAC transcription factors, as well as structural genes and phytohormone-related genes, in modulating anthocyanin metabolism during postharvest color changes in raspberries. The findings provide valuable insights for optimizing postharvest fruit storage conditions and enhancing fruit quality.

Keywords: anthocyanin; color variation; metabolome; raspberry; transcriptome.

Figure 1

Color and anthocyanin content of raspberry fruit. (A) Color variation of postharvest raspberry fruit during storage. RBT-1 to RBT-5: white to deep red fruits. (B) Total anthocyanin content in RBT-1 and RBT-5. (C) L* values in the RBT-1 to RBT-5 samples. (D) a* values in the RBT-1 to RBT-5 samples. (C–E) b* values in the RBT-1 to RBT-5 samples. Values represent the mean ± standard deviation (SD) of three biological replicates. The bar chart with different lowercase letters indicates that the difference is significant (p < 0.05).

Figure 2

Analysis of metabolome. (A) PCA score plots of raspberries at different color stages. The RBT-1–1 to RBT-1-3, RBT-2–1 to RBT-2-3, RBT-3–1 to RBT-3-3, RBT-4–1 to RBT-4-3, and RBT-5–1 to RBT-5–3 represents three biological replicates for five raspberry color stages. (B) Anthocyanin quantity. (C) Heat maps of the 43 anthocyanin metabolites. (D) The content of 9 DAMs in RBT-1, RBT-3 and RBT-5. (E) log2FC values of the nine DAMs in the two comparison groups. Red represents the RBT-3 vs. RBT-1 comparison group, and green represents the RBT-5 vs. RBT-1 comparison group.

Figure 3

Analysis of differentially expressed genes in the transcriptome. (A) PCA score plot of 15 transcriptome samples. (B) DEGs quantity in the four groups. The thresholds is |log2FC| ≥ 1.0 and padj < 0.05. (C) Venn plot of DEGs at different color stages. (D) KEGG enrichment analysis of four groups. The q values range from 0-1. The smaller the value, the more significant the enrichment.

Figure 4

Differentially expressed structural gene regulatory network of the anthocyanin biosynthetic pathway in raspberry. Expression levels from low to high are represented as brown to blue. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3’H, flavonoid 3’-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanin synthase; UFGT, flavonoid 3-O-glucosyltransferase.

Figure 5

Heatmaps of differentially expressed TFs associated with anthocyanin biosynthesis pathways. Expression levels from low to high are represented as purple to orange.

Figure 6

Heatmaps of DEGs participating in plant hormone metabolism. Expression levels from low to high are represented as orange to purple.

Figure 7

Weighted gene co-expression network analysis. (A) Cluster tree diagram of DEGs identified by WGCNA. (B) Heatmap of the correlation between DAMs and modules. (C) Expression trends of DEGs in the turquoise module. (D) The expression trend of DEGs in pink module. (E) Expression trends of DEGs in the blue module. (F) Expression trends of DEGs in the yellow module.

Figure 8

Network diagram of anthocyanin synthesis-related genes and metabolites in raspberry fruit. (A) Correlation network diagram of five DAMs and candidate DEGs. (B) Correlation network diagram of cyanidin-3-O-sophoroside and candidate DEGs. The square is described as a plant hormone signal gene, triangle is described as a structural gene, and hexagon is described as a TFs.. Cy-O-sop, Cyanidin-3-O-sophoroside; Cy-gen, Cyanidin-3-gentiobioside; Pe-O-dig, Pelargonidin-3,5-O-diglucoside; Cy-O-sam, Cyanidin-3-O-sambubioside; Pe-O-sam, Pelargonidin-3-O-sambubioside.

Figure 9

RT-qPCR validation of nine key genes related to anthocyanin content in color-variant raspberry during storage. Error bars indicate the standard deviation (SD) of three technical repeats.