Integrative Analysis of Metabolomics and Transcriptomics Reveals Molecular Mechanisms of Anthocyanin Metabolism in the Zikui Tea Plant (Camellia sinensis cv. Zikui)

Abstract

In this study, we performed an association analysis of metabolomics and transcriptomics to reveal the anthocyanin biosynthesis mechanism in a new purple-leaf tea cultivar Zikui (Camellia sinensis cv. Zikui) (ZK). Three glycosylated anthocyanins were identified, including petunidin 3-O-glucoside, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside, and their contents were the highest in ZK leaves at 15 days. This is the first report on petunidin 3-O-glucoside in purple-leaf tea. Integrated analysis of the transcriptome and metabolome identified eleven dependent transcription factors, among which CsMYB90 had strong correlations with petunidin 3-O-glucoside, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside (PCC > 0.8). Furthermore, we also identified key correlated structural genes, including two positively correlated F3’H (flavonoid-3′-hydroxylase) genes, two positively correlated ANS (anthocyanin synthase) genes, and three negatively correlated PPO (polyphenol oxidase) genes. Overexpression of CsMYB90 in tobacco resulted in dark-purple transgenic calluses. These results showed that the increased accumulation of three anthocyanins in ZK may promote purple-leaf coloration because of changes in the expression levels of genes, including CsMYB90, F3’Hs, ANSs, and PPOs. These findings reveal new insight into the molecular mechanism of anthocyanin biosynthesis in purple-leaf tea plants and provide a series of candidate genes for the breeding of anthocyanin-rich cultivars.

Keywords: anthocyanin metabolism; association analysis; metabolomics; purple-leaf tea plant; transcriptomics; ‘Zikui’.

Figure 1

Observation of the purple phenotype and comparison of anthocyanin content changes in Zikui (ZK) and N61 tea cultivars in 5-, 10-, 15-, 30-, and 45-day-old leaves. (A) Observation of the purple phenotype, bar = 1 cm. (B) Anthocyanin content changes.

Figure 2

Analysis of differential metabolite components and relative content of key metabolites in the Zikui (ZK) and N61 cultivars. (A) Numbers of differential metabolites. (B) Relative content of key metabolites in ZK and N61 at 15 and 45 days, presented as dot whisker plots (n = 3 independent biological samples; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Figure 3

Analysis of differentially expressed genes between the 15- and 45-day stages of the Zikui (ZK) and N61 cultivars. (A) Numbers of differentially expressed genes. (B) UpSet graphs.

Figure 4

Correlation analysis between genes and key metabolites of purple-leaf formation in Zikui tea. (A) Mapping of key metabolites and strongly correlated genes. mws0997, mws1048, and pmb0550 in purple are the metabolites petunidin 3-O-glucoside, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside, respectively. mws1293, mws1295, and mws1296 in pink are the metabolites theaflavin, theaflavin-3’-gallate, and theaflavine-3, 3’-digallate, respectively. Yellow represents structural genes, and light blue represents transcription factors. Unannotated genes that are highly associated with these metabolites are indicated in orange. The thickness of the line indicates the strength of the correlation. (B) Correlation analysis of 18 key genes and key metabolites. (C) Heatmap showing the expression levels of 18 key genes, n = 3 independent biological samples.

Figure 5

Transcriptional profiling of genes involved in the phenylpropane and flavonoid biosynthesis pathways in Zikui (ZK) and N61 tea cultivars. Grids with a purple color scale from light to dark represent fragments per kilobase of exon per million fragments mapped (FPKM) values of 0–10, 10–20, 20–40, 40–80, 80–160, and 160–320. Grids with a red color scale from light to dark represent FPKM values of 0–10, 10–20, 20–40, 40–80, 80–160, 160–320, 320–640, 640–1280, 1280–2560, and more than 2560. PAL, phenylalanine ammonia-lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; UFGT, UDP glucose-flavonoid 3-O-glcosyl-transferase; LAR, leucocyanidin reductase.

Figure 6

Expression of representative genes in Zikui (ZK) and N61 tea cultivars validated by quantitative real-time polymerase chain reaction (qRT-PCR). Presented as dot and whisker plots (n = 3 independent biological samples; ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Figure 7

Overexpression of CsMYB90 resulted in visible dark-purple coloration in the calluses of transgenic tobacco. Bar = 1 cm.