Metabolome and Transcriptome Sequencing Analysis Reveals Anthocyanin Metabolism in Pink Flowers of Anthocyanin-Rich Tea (Camellia sinensis)

Abstract

Almost all flowers of the tea plant (Camellia sinensis) are white, which has caused few researchers to pay attention to anthocyanin accumulation and color changing in tea flowers. A new purple-leaf cultivar, Baitang purple tea (BTP) was discovered in the Baitang Mountains of Guangdong, whose flowers are naturally pink, and can provide an opportunity to understand anthocyanin metabolic networks and flower color development in tea flowers. In the present study, twelve anthocyanin components were identified in the pink tea flowers, namely cyanidin O-syringic acid, petunidin 3-O-glucoside, pelargonidin 3-O-beta-d-glucoside, which marks the first time these compounds have been found in the tea flowers. The presence of these anthocyanins seem most likely to be the reason for the pink coloration of the flowers. Twenty-one differentially expressed genes (DEGs) involved in anthocyanin pathway were identified using KEGG pathway functional enrichment, and ten of these DEG's screened using venn and KEGG functional enrichment analysis during five subsequent stages of flower development. By comparing DEGs and their expression levels across multiple flower development stages, we found that anthocyanin biosynthesis and accumulation in BTP flowers mainly occurred between the third and fourth stages (BTP3 to BTP4). Particularly, during the period of peak anthocyanin synthesis 17 structural genes were upregulated, and four structural genes were downregulated only. Ultimately, eight critical genes were identified using weighted gene co-expression network analysis (WGCNA), which were found to have direct impact on biosynthesis and accumulation of three flavonoid compounds, namely cyanidin 3-O-glucoside, petunidin 3-O-glucoside and epicatechin gallate. These results provide useful information about the molecular mechanisms of coloration in rare pink tea flower of anthocyanin-rich tea, enriching the gene resource and guiding further research on anthocyanin accumulation in purple tea.

Keywords: Camellia sinensis (L.); WGCNA; anthocyanin biosynthesis; anthocyanin-rich cultivar; flower development; metabolome; transcriptome.

Figure 1

PCA scores plot of different floral development stages by UPLC-ESI-MS/MS metabolite analysis. PC1, principal component 1; PC2, principal component 2. Explained variants PC1: 77.1%, PC2: 9.7%.

Figure 2

OPLS-DA score plots demonstrating the clustering pattern of metabolites in subsequent development stages of tea flowers. X-axes represent PC1, Y-axes represent PC2; RMSEE represent Root Mean Square Error of Estimation. (A) BTP1 vs BPT2 group (R2Y  =  1, Q2Y  =  0.992, RMSEE  =  0.002 ); (B) BTP2 vs BPT3 group (R2Y  =  1, Q2Y = 0.959, RMSEE  =  0.008); (C) BTP3 vs BPT4 group (R2Y  =  0.999, Q2Y  =  0.938,RMSEE = 0.02); (D) BTP4 vs BPT5 group (R2Y  =  1, Q2Y  =  0.975,RMSEE  =  0.002).

Figure 3

GO classification of unigenes from pink Camellia sinensis flower.

Figure 4

Classification of unigenes from pink Camellia sinensis flower.

Figure 5

Differentially expressed genes at various stages of BTP flower development (A) DEGs among BTP1 vs. BTP2, BTP1 vs. BTP3, BTP1 vs. BTP4, and BTP1 vs. BTP5. Blue: up–regulated expression of DEGs, Red: down–regulated. (B) Venn Diagram of DEGs between 4 groups.

Figure 5

Differentially expressed genes at various stages of BTP flower development (A) DEGs among BTP1 vs. BTP2, BTP1 vs. BTP3, BTP1 vs. BTP4, and BTP1 vs. BTP5. Blue: up–regulated expression of DEGs, Red: down–regulated. (B) Venn Diagram of DEGs between 4 groups.

Figure 6

KEGG enrichment analysis of DEGs in different tea development stages.

Figure 7

Key structural genes and their expression level of involved in anthocyanin biosynthesis pathway. (A) the number of DEGs involved in anthocyanin biosynthetic pathway. (B) The heatmap of key structural genes and expression level. The color scale represents the log2 values (FPKM) of the tea flower during the BTP1, BTP2, BTP3, BTP4, and BTP5 stages (from left to right).

Figure 7

Key structural genes and their expression level of involved in anthocyanin biosynthesis pathway. (A) the number of DEGs involved in anthocyanin biosynthetic pathway. (B) The heatmap of key structural genes and expression level. The color scale represents the log2 values (FPKM) of the tea flower during the BTP1, BTP2, BTP3, BTP4, and BTP5 stages (from left to right).

Figure 8

Co-expression and crucial gene screening. (A) Clustering results of co-expression modules. Genes in modules are marked with different colors (blue, yellow, brown and turquoise), with grey color representing no genes in any modules. (B) Clustering dendrogram. (C) Relationships of module and seven different anthocyanins found in BTP flowers.

Figure 8

Co-expression and crucial gene screening. (A) Clustering results of co-expression modules. Genes in modules are marked with different colors (blue, yellow, brown and turquoise), with grey color representing no genes in any modules. (B) Clustering dendrogram. (C) Relationships of module and seven different anthocyanins found in BTP flowers.

Figure 9

Expression analysis for structural genes involved in anthocyanin accumulation. (A) Expression level analysis of key structural genes involved in anthocyanin biosynthesis in pink tea flower development stages. (B) Correlation analysis based on RNA-seq and qRT-PCR data.

Figure 9

Expression analysis for structural genes involved in anthocyanin accumulation. (A) Expression level analysis of key structural genes involved in anthocyanin biosynthesis in pink tea flower development stages. (B) Correlation analysis based on RNA-seq and qRT-PCR data.