Transcriptome analysis of bagging-treated red Chinese sand pear peels reveals light-responsive pathway functions in anthocyanin accumulation

Abstract

Bagging is an efficient method to improve fruit colour development. This work reported a transcriptome analysis using bagging-treated red Chinese sand pear peels. In total, 8,870 differentially expressed genes were further analysed by a weighted gene co-expression network analysis and early-, middle- and late light-responsive genes were identified. An annotation analysis revealed several pathways involved in the different responsive stages. The presence of LONG HYPOCOTLY 5, CRY-DASH and a CONSTANS-like transcription factors among the early light-responsive genes indicated the pivotal role of light, especially blue light, in the biological changes that occurred after bag removal. Other light-responsive transcription factors were also identified from the three light-responsive stages. In addition, the light-responsive pattern of anthocyanin biosynthetic genes differed among the biosynthetic steps. Although yeast-one hybrid assay showed that most of the structural genes were regulated by PpMYB10, their different temporal expressive pattern suggested that besides PpMYB10, other light-responsive transcriptional factors were also involved in the regulation of anthocyanin biosynthesis. In summary, our transcriptome analysis provides knowledge of the transcriptional regulatory network operating during light responses, which results in anthocyanin accumulation and other significant physiological changes in red Chinese sand pear peels after bag removal.

Figure 1

Changes in pigmentation of fruit peel of red Chinese sand pear in bagged and bag-removed samples. (a) Photos for bag-removed pear ‘Meirensu’. (b) Content changes of anthocyanin (up), carotenoid (middle) and chlorophyll (down) in fruit peel after bag removal. The labels of the samples were displayed as “hours after bag-removal” (h). The graphs showed the average values from three independently sampled fruits as biological replicates. Error bars are the standard deviation. Asterisk indicates p < 0.05.

Figure 2

Comparison of the differential expressed genes of each pairs. The labels of the samples are displayed as “hours after bag-removal” (H) or “hours without bag-removal” (HC) Note that bag removal significantly changes the gene expression pattern in the fruit peel, while genes of the control had an expression pattern similar to the 0 h sample.

Figure 3

Weighted gene co-expression network analysis (WGCNA) of DEGs identified from ‘Meirensu’ pear peel after bag removal. (a) Hierarchical cluster tree showing 17 modules of co-expressed genes. Each of the 8,870 DEGs is represented by a tree leaf and each of the modules by a major tree branch. The lower panel shows modules in designated colours. (b) Module–trait correlations and corresponding p-values (in parentheses). The left panel shows the 17 modules and the number of member genes. The colour scale on the right shows module–trait correlations from −1 (blue) to 1 (red). The left panel “Anthocyanin” represents anthocyanin biosynthesis as a trait. The middle panel “UFGT2” represents the expression changes of PpUFGT2, which encodes the enzyme that catalyses the last step in anthocyanin biosynthesis, as a trait. The right panel “MYB10” represents the expression changes of PpMYB10, which is the key transcriptional factor activating anthocyanin biosynthesis, as a trait. (c) Cytoscape representation of co-expressed genes with edge weights ≥0.40 in module ‘blue’ and ‘midnight blue’. (d) RNA-Seq expression patterns of PpGST, Pp UFGT2 and PpMYB10.

Figure 4

Heat maps showing the expression patterns of modules from the light-responsive genes.

Figure 5

Analysis of the early-responsive genes. (a) Gene ontology enrichment analysis of early-responsive genes. (b) qPCR confirmation of the expression patterns of selected genes.

Figure 6

Transcription factor analysis of middle- (a) and late- (b) responsive genes. The numbers of up-regulated genes and down-regulated genes comparing to 0 h were showed with blue (up) and green (down), respectively.

Figure 7

Categorisation of genes belonging to the phenylalanine pathway. Structural genes within the phenylalanine pathway were assigned to different light-responsive gene sets.

Figure 8

Direct binding of PpMYB10 on the promoter of anthocyanin biosynthetic structural genes (a) and PpMYB10 itself (b) of ‘Meirensu’ in yeast-one hybrid system. The tri-angle marked the conserved MYB binding domain predicted using PlantCare.

Figure 9

Putative model for bag removal-induced anthocyanin biosynthesis in ‘Meirensu’ pear peels. The phytohormone and other pathways based on the gene ontology enrichment analysis.