Integrated transcriptome and metabolome profiling of Camellia reticulata reveal mechanisms of flower color differentiation

Abstract

Camellia reticulata (Lindl.) is an important ornamental plant in China. Long-term natural or artificial selections have resulted in diverse phenotypes, especially for flower colors. Modulating flower colors can enhance the visual appeal and economic value in ornamental plants. In this study, we investigated the molecular mechanisms underlying flower color differentiation in C. reticulata. We performed a combined transcriptome and metabolome analysis of the petals of a popular variety C. reticulata (HHYC) (red), and its two cultivars "Xuejiao" (XJ) (pink) and "Tongzimian" (TZM) (white). Targeted metabolome profiling identified 310 flavonoid compounds of which 18 anthocyanins were differentially accumulated among the three samples with an accumulation pattern of HHYC > XJ > TZM. Likewise, transcriptome analysis showed that carotenoid and anthocyanin biosynthetic structural genes were mostly expressed in order of HHYC > XJ > TZM. Two genes (gene-LOC114287745765 and gene-LOC114289234) encoding for anthocyanidin 3-O-glucosyltransferase are predicted to be responsible for red coloration in HHYC and XJ. We also detected 42 MYB and 29 bHLH transcription factors as key regulators of anthocyanin-structural genes. Overall, this work showed that flavonoids, particularly anthocyanins contents are the major determinants of flower color differentiation among the 3 C. reticulata samples. In addition, the main regulatory and structural genes modulating anthocyanin contents in C. reticulata have been unveiled. Our results will help in the development of Camellia varieties with specific flower color and quality.

Keywords: Camellia reticulata; anthocyanins; carotenoids; flower; omics.

FIGURE 1

Different flower colors among three Camellia reticulata. (A) C. reticulata (HHYC), (B) C. reticulata ‘Tongzimian’ (TZM), (C) C. reticulata ‘Xuejiao’ (XJ).

FIGURE 2

Quantification of fragments per kilobase of exon per million fragments mapped (FPKM) among the nine samples (3 contrasting flowers × 3 biological replicates). (A). FPKM distribution among the nine samples. (B). Principal components analysis of three contrasting flowers of C. reticulata (HHYC1-3), C. reticulata ‘Xuejiao’ (XJ1-3) and C. reticulata ‘Tongzimian’ (TZM1-3).

FIGURE 3

Differentially expressed genes in the three contrasting flowers of C. reticulata (HHYC), C. reticulata ‘Xuejiao’ (XJ) and C. reticulata ‘Tongzimian’ (TZM). (A). Hierarchical clustering heatmap based on fragments per kilobase of exon per million fragments mapped of differentially expressed genes (DEGs) of the three samples (HHYC, XJ and TZM) based on triplicate biological repeats. The red, blue and green colors on column cluster (shown right hand side) represent HHYC, XJ and TZM, respectively. (B). Extent of regulation of DEGs. (C). Venn diagram of DEGs among the three pairwise group comparisons (n = number of DEGs detected).

FIGURE 4

Heatmap clustering of thirty-eight structural genes involved in carotenoid biosynthesis among the three contrasting flowers of C. reticulata (HHYC), C. reticulata ‘Xuejiao’ (XJ) and C. reticulata ‘Tongzimian’ (TZM). The expression levels of the genes were log2 transformed from averages of the three biological repeats from each genotype and the transformed data were used to draw the heatmap. Color diagram at the right side shows the intensity of the gene expression.

FIGURE 5

Profiles of metabolites accumulated among the 3 from petals of contrasting flowers of C. reticulata (HHYC; red colored), C. reticulata ‘Xuejiao’ (XJ; pink colored) and C. reticulata ‘Tongzimian’ (TZM; white colored). (A). Principal component analysis based on metabolites normalized ion intensities. Mix samples represent equal proportion of samples from HHYC, XJ and TZM. Metabolome profiling was done in triplicates for each genotype (HHYC-1 to -3, XJ-1 to -3, TZM-1 to -3 and mix-1 to -3). (B). Hierarchical clustering heatmap based on metabolites normalized ion intensities. The red, blue and green colors on column cluster (shown righthand side) represent HHYC, XJ and TZM, respectively. The ion intensity of each metabolite are shown green color represents lowly-accumulated, and red color represents highly-accumulated metabolites.

FIGURE 6

Schematic diagram of anthocyanins biosynthetic pathways responsible for color differentiation among C. reticulata (HHYC), C. reticulata ‘Xuejiao’ (XJ) and C. reticulata ‘Tongzimian’ (TZM). (A). Heatmap of four anthocyanidin 3-O-glucosyltransferase [EC:2.4.1.115] (BZI) encoded genes. (B). Heatmap of seven differentially accumulated metabolites. Metabolites with hollow circles were not detected in this study, while those in purple circles were detected in this study. Heatmaps were produced with log2 transformed data. Color diagram at the right side shows the intensity of the metabolite accumulation or gene expression.

FIGURE 7

Pearson correlation network diagram of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) abundance in pairwise comparison of C. reticulata (HHYC), C. reticulata ‘Xuejiao’ (XJ) and C. reticulata ‘Tongzimian’ (TZM). (A). HHYC_vs_XJ. (B). HHYC_vs_TZM. (C). Heatmap clustering of seven metabolites based on log2 transformed of ion intensities. The metabolites pme3392 (pelargonidin-3-O-glucoside), pmb0550 (cyanidin-3-O-glucoside (kuromanin)), pmf0203 (peonidin-3-O-glucoside), pmb0542 (cyanidin-3-O-(6″-O-malonyl) glucoside), pme1773 (cyanidin-3-O-rutinoside (keracyanin)), pme1777 (cyanidin-3,5-O-diglucoside (cyanin)) and pmf0116 (delphinidin-3,5-di-O-glucoside). The DEGs in the network (gene-LOC114285765 and gene-LOC114289234 encode for anthocyanidin 3-O-glucosyltransferase [EC:2.4.1.115] designated BZI in Anthocyanin biosynthetic pathway in Supplementary Figure S3). Color diagram at the right side shows the intensity of the metabolite accumulation.