Study on the dynamics of fruits color related genes and metabolites during the fruit development of Zanthoxylum bungeanum 'Hanyuan'

Abstract

Background: Anthocyanins play a crucial role in determining the coloration of plant fruits and are essential for enhancing their economic traits. However, studies on anthocyanin biosynthesis in Zanthoxylum bungeanum 'Hanyuan' fruit have yet to be reported.

Results: In this study, the patterns of anthocyanin synthesis in fruit through phenotypic analysis and total anthocyanin measurements across 13 development stages were traced. The results indicated that the pericarp of Z. bungeanum 'Hanyuan' exhibited a gradual transition in color from light red to dark red between 64 d AF and 100 d AF, with the highest total anthocyanin content recorded at 64 d AF. Based on this conclusion, five key stages were selected for the transcriptome and anthocyanin-targeted metabolome analysis to further elucidate the molecular mechanisms underlying anthocyanin synthesis. Metabolomic analysis identified eight metabolites that influence the formation of red color in fruit: Cyanidin-3-O-rutinoside, cyanidin-3-O-xyloside, naringenin, peonidin-3-O-rutinoside, cyanidin-3-O-(6-O-malonyl-beta-D-glucoside), petunidin-3-O-sambubioside, petunidin-3-O-(6-O-p-coumaroyl)-glucoside, and delphinidin-3-O-(6-O-malonyl-beta-D-glucoside). These metabolites exhibited the highest concentrations at 64 d AF, 86 d AF, and 100 d AF. Transcriptome analysis, WGCNA analysis, and correlation analysis indicate that ZbFLS11 and ZbCYP98A32 positively regulate the biosynthesis of cyanidin-3-O-xyloside, petunidin-3-O-(6-O-p-coumaroyl) glucoside, and petunidin-3-O-sambubioside. Zb4CL1 exhibits a positive correlation with petunidin-3-O-(6-O-p-coumaroyl)-glucoside and naringenin. Additionally, the upregulation of five genes (ZbCHS1, ZbCHS2, ZbCHS3, ZbCHS4, and ZbCHS5), ZbC4H2, and ZbC4H3 expressions at 64 d AF and 86 d AF was associated with the accumulation of delphinidin-3-O-(6-O-malonyl-beta-D-glucoside) during the development stages of Z. bungeanum 'Hanyuan' fruit. Furthermore, it was found that MYB113 interact with the structural genes Zb4CL1, ZbCYP98A32, and ZbFLS11, thereby regulating the biosynthesis of petunidin-3-O-(6-O-p-coumaroyl)-glucoside. Additionally, bHLH6 exhibits a positive regulatory relationship with the five genes (ZbCHS1, ZbCHS2, ZbCHS3, ZbCHS4, and ZbCHS5), ZbC4H2, and ZbC4H3, which influence the biosynthesis of delphinidin-3-O-(6-O-malonyl-beta-D-glucoside).

Conclusion: In summary, the findings of this study elucidate the molecular mechanisms underlying color development in Z. bungeanum 'Hanyuan' fruit across various development stages. This research offers valuable insights for future investigations into the intricate molecular network governing anthocyanin biosynthesis in Z. bungeanum 'Hanyuan' fruit.

Keywords: Zanthoxylum bungeanum ‘Hanyuan’; Anthocyanin; Metabolomic; Transcription factors; Transcriptomic; Weighted gene co-expression network analysis.

Fig. 1

Coloration and pigment content in fruit during the development. a Appearance of fruit during the development, bar means 1 mm; b Total anthocyanin content in fruit at the different stages; c Carotenoid contents in fruit at the different stages; d Chlorophyll contents in fruit at the different stages. Note: Lowercase letters indicate significant differences between treatments (P < 0.05)

Fig. 2

Analysis of anthocyanin metabolites at the different stages. a CV distribution of samples at each stage. The vertical axis represents the proportion of substances with values less than the CV, relative to the total number of substances. Each color represented a different stage of the sample group. The proportion of QC samples with CV values below 0.3 exceeded 80%, which indicates the stability of the experimental data. The proportion of QC samples with CV values below 0.2 exceeded 80%, which indicates a high level of stability in the experimental data; b PCA of anthocyanins in samples of 15 different stages; c Intra-group correlation diagrams of 15 samples in the five periods; d Classification and proportion of 60 anthocyanins in fruit

Fig. 3

Comparison of anthocyanin metabolites at the different stages. a Venn diagram; b Quantity of differential metabolites at 23 d AF, 39 d AF, 64 d AF, 86 d AF and 100 d AF, red color indicates upregulation of differential metabolites, and green color represents downregulated differential metabolites; c 39 d AF vs. 23 d AF; d 39 d AF vs. 23 d AF; e 86 d AF vs. 23 d AF; f 100 d AF vs. 23 d AF, the x-coordinate indicates the rich factor corresponding to each pathway. The y-coordinate represents the pathway names associated with differential metabolites enrichment analysis. The dot color indicates the P-value, and the dot size represents the extent of enrichment of differential metabolites

Fig. 4

Differential analysis of 20 DAAs at the different development stages. a-d Relationships between up-and down-regulation of 20 differential anthocyanins in the different groups. Pink color represents the upregulation of differentially expressed metabolites and green represents the down-regulation of differentially expressed metabolites. The red text represents the anthocyanins shared by the four comparison groups. The yellow text represents anthocyanins shared at 64 d AF vs. 23 d AF, 86 d AF vs. 23 d AF, and 100 d AF vs. 23 d AF comparison groups. The purple text represents anthocyanins up-regulated at 64 d AF vs. 23 d AF, 86 d AF vs. 23 d AF, and 100 d AF vs. 23 d AF

Fig. 5

Volcanic map of differential genes. a-d Volcano plot of DEGs at 39 d AF vs. 23 d AF, 64 d AF vs. 23 d AF, 86 d AF vs. 23 d AF, and 100 d AF vs. 23 d AF, respectively. Pink spots represent up-regulated genes in four comparison groups, green spots represent down-regulated genes in the four comparison groups, and gray spots represent genes with no significant change in the four comparison groups

Fig. 6

KEGG analysis of sample DEGs at the different development stages. a-d Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment bubble diagrams of DEGs at 39 d AF vs. 23 d AF, 64 d AF vs. 23 d AF, 86 d AF vs. 23 d AF, and 100 d AF vs. 23 d AF, respectively. The size of the bubble indicates the number of differentially expressed genes enriched in this KEGG pathway; the color of the bubble indicates the P-value

Fig. 7

Anthocyanin biosynthesis pathway. The red and violet blocks represent DEGs

Fig. 8

Correlation network diagram of the eight DAAs and eighteen key candidate genes involved in the anthocyanin biosynthesis. Orange circles represent enzyme-encoding genes, violet circle boxes represent DAAs, orange solid lines indicate positive correlations between the genes and anthocyanin metabolites, and blue dashed lines indicate negative correlations between the genes and anthocyanin metabolites

Fig. 9

Correlation network diagram of sixty key candidate genes for anthocyanin biosynthesis associated with the eleven MYBs and fourteen bHLHs TFs. Triangles represent TFs, circles represent the key structural genes involved in anthocyanin biosynthesis, red solid lines represent a positive correlation between the two, and purple dashed lines represent a negative correlation between the two

Fig. 10

WGCNA results of overlapping differential genes and differentially accumulated metabolites in the four control groups. a Clustering dendrogram results showing the five expression modules, each marked with a different color; b Module-trait relationships between 8 DAAs and genes in the four control groups, the value in each box indicates the Pearson correlation coefficient between the modules containing DAAs, and the number in each bracket indicates the P-value. The color scale on the right side represents the correlation degree between the modules and DAAs, and the red color represents high correlation; c, d The co-expressed genes in fruit at 23 d AF, 39 d AF, 64 d AF, 86 d AF, and 100 d AF

Fig. 11

Network diagram of fruit development in Z. bungeanum ‘Hanyuan’. In the fruit development of Z. bungeanum ‘Hanyuan’, MYB113 and bHLH6 were significantly upregulated. The interaction between MYB113 and bHLH6 was noteworthy. The MYB113 binds to the promoters of genes involved in the anthocyanin biosynthetic pathway, thereby promoting anthocyanin biosynthesis. The accumulation of anthocyanins results in the red coloration of Z. bungeanum ‘Hanyuan’. However, the specific regulatory mechanisms governing the MYB113 warrant further investigation