Integrating metabolite and transcriptome analysis revealed the different mechanisms of characteristic compound biosynthesis and transcriptional regulation in tea flowers

Abstract

The flowers of tea plants (Camellia sinensis), as well as tea leaves, contain abundant secondary metabolites and are big potential resources for the extraction of bioactive compounds or preparation of functional foods. However, little is known about the biosynthesis and transcriptional regulation mechanisms of those metabolites in tea flowers, such as terpenoid, flavonol, catechins, caffeine, and theanine. This study finely integrated target and nontarget metabolism analyses to explore the metabolic feature of developing tea flowers. Tea flowers accumulated more abundant terpenoid compounds than young leaves. The transcriptome data of developing flowers and leaves showed that a higher expression level of later genes of terpenoid biosynthesis pathway, such as Terpene synthases gene family, in tea flowers was the candidate reason of the more abundant terpenoid compounds than in tea leaves. Differently, even though flavonol and catechin profiling between tea flowers and leaves was similar, the gene family members of flavonoid biosynthesis were selectively expressed by tea flowers and tea leaves. Transcriptome and phylogenetic analyses indicated that the regulatory mechanism of flavonol biosynthesis was perhaps different between tea flowers and leaves. However, the regulatory mechanism of catechin biosynthesis was perhaps similar between tea flowers and leaves. This study not only provides a global vision of metabolism and transcriptome in tea flowers but also uncovered the different mechanisms of biosynthesis and transcriptional regulation of those important compounds.

Keywords: metabolism; regulation mechanism; tea flowers; transcription factor; transcriptome.

Figure 1

Potential application of tea flowers in tea processing. (A) Appearance of tea flower tea products. TFT, tea flower tea; LYT, large-leaf yellow tea. (B) Soup color from mixed tea products with different ratios between TFT and LYT. The ratio number between TFT and LYT is listed on top of each figure. (C) Effect of TFT on taste attributes. (D) Venn diagram showing the volatile compounds of tea flowers contributing to tea aroma.

Figure 2

Metabolic analysis of tea flowers and leaves. (A) GC-MS total ion current of volatiles in tea flowers and leaves. (B) Venn diagram showing the variation of volatile compounds in tea flowers and leaves. (C, D) HPLC chromatographs of catechins and caffeine (C) and flavonols (D) in tea flowers and leaves. The compound names are listed on top of the peaks. EGCG, epigallocatechin gallate; ECG, epicatechin gallate; K-7-0-Glu, kaempferol-7-O-glucoside; K-3-0-Glu, kaempferol-3-O-glucoside; Q-3-0-Glu, quercetin-3-O-glucoside; M-3-0-Glu, myricetin 3-O-glucoside. (E) Phenotype of developing tea flowers. F1, flower stage 1; F2, flower stage 2; F3, flower stage 3; F4, flower stage 4; F5, flower stage 5; F6, flower stage 6. (F, G) Contents of caffeine and catechins in developing tea flowers (F) and different parts of tea flowers (G). (H, I) Contents of flavonols in developing tea flowers (H) and different parts of tea flowers (I). All data are from at least three biological replicates and are expressed as mean ± SD.

Figure 3

Nontarget metabolic analysis of tea flowers and leaves. (A) Principal component analysis of tea flower and leaf samples. PC1, the first principal component. PC2, the second principal component. For each tissue samples, six biological replicates were prepared. (B) Volcano plots of differential metabolites in tea flower and leaf samples. (C) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of different metabolites in tea flower and leaf samples. (D) Five most abundant metabolites in tea flowers. (E) Differences in key metabolites between tea flowers and leaves.

Figure 4

Expression analysis of TPS gene family in tea developing flowers and leaves. (A) Heat map expression analysis of TPS genes involved in the terpenoid process in tea developing flowers and leaves. F1, tea flower stage 1; F2, tea flower stage 2; F3, tea flower stage 3; F4, tea flower stage 4; F5, tea flower stage 5; F6, tea flower stage 6; ST, stamen; PE, petal; RE, receptacle; L1, first tea leaf; L2, second tea leaf; L3, third tea leaf; L4, fourth tea leaf; L5, fifth tea leaf; and L6, sixth tea leaf; S1, internode between the first leaf and second leaf; S2, internode between the second leaf and third leaf; S3, internode between the third leaf and fourth leaf; S4, internode between the fourth leaf and fifth leaf; Mono_syn, monoterpene synthase; Sesqui_syn, sesquiterpene synthase; Di_syn, diterpene synthase. The expression level [log10(FPKM)] of each gene is shown in the heat map boxes. (B) Cumulative expression analysis of the TPS family genes in tea developing flowers and leaves.

Figure 5

Expression analysis of gene families involved in flavonoid biosynthesis in tea developing flowers and leaves. (A, B) Heat map expression analysis of CHS genes in tea developing flowers (A) and leaves (B). (C, D) Heat map expression analysis of FLS genes in tea developing flowers (C) and leaves (D). (E) Relative expression level of gene family members in different tissues of tea plants. F1, tea flower stage 1; F2, tea flower stage 2; F3, tea flower stage 3; F4, tea flower stage 4; F5, tea flower stage 5; F6, tea flower stage 6; ST, stamen; PE, petal; RE, receptacle; L1, first tea leaf; L2, second tea leaf; L3, third tea leaf; L4, fourth tea leaf; L5, fifth tea leaf; L6, sixth tea leaf; S1, internode between the first leaf and second leaf; S2, internode between the second leaf and third leaf; S3, internode between the third leaf and fourth leaf; S4, internode between the fourth leaf and fifth leaf; PAL, phenylalanine ammonialyase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol reductase; FLS, flavonol synthase. The expression level [log10(FPKM)] of each gene is shown in the heat map boxes.

Figure 6

Transcriptional regulatory mechanism of flavonol and catechin biosynthesis in tea flowers. (A, B) Pearson correlation analysis of the expression of FLS genes and the expression of three flavonol MYB regulator genes in developing leaves (A) and developing flowers (B). The sizes of circles indicate the degree of correlation. Green indicates a positive correlation, and red indicates a negative correlation. (C) Phylogenetic tree of tea MYBs with others related to flavonol biosynthesis regulation in flowers. The numbers at the nodes indicate the bootstrap value with 1,000 replicates. The red MYB genes highlight their corresponding homolog genes in tea plants. (D) Heat map expression analysis of candidate MYB genes in different tea tissues. AB, apical bud; FL, flower; FR, fruit; YL, young leaf; ML, mature leaf; OL, old leaf, RT, root, ST, stem. The expression level [log10(FPKM)] of each gene is shown in the heat map boxes. (E) Expression level analysis of candidate MYB genes regulating flavonol biosynthesis in tea developing flowers. (F, G) Pearson correlation analysis of the expression of ANR and LAR genes and the expression of catechin regulator genes in developing leaves (F) and developing flowers (G).

Figure 7

Caffeine and theanine biosynthesis in tea flowers. (A, B) Main biosynthetic route toward caffeine biosynthesis and degradation (A) and theanine biosynthesis (B) in tea developing flowers and leaves. AMP, adenosine monophosphate; IMP, inosine monophosphate; XMP, xanthosine monophosphate; Anase, adenosine nucleosidase; APRT, adenine phosphoribosyltransferase; AMPD, AMP deaminase; IMPDH, IMP dehydrogenase; 5′-Nase, 5′-nucleotidase; 7-NMT, 7-methylxanthosine synthase; N-MeNase, N-methylnucleotidase; MXMT, theobromine synthase; TCS, tea caffeine synthase; XO, xanthine oxidase; XDH, xanthine dehydrogenase; ALN, allantoinase; ALLC, allantoicase; GS, glutamine synthetase; GOGAT, glutamate synthase; GDH, glutamate dehydrogenase; AlaDC, alanine decarboxylase; GGT, γ-glutamyltranspeptidase; TSI, theanine synthetase. The log 10 (expression levels, TPM) of the genes is represented by a heat map.