Identification, Characterization and Expression Profiling of the RS Gene Family during the Withering Process of White Tea in the Tea Plant (Camellia sinensis) Reveal the Transcriptional Regulation of CsRS8

Abstract

Raffinose synthetase (RS) is a key enzyme in the process of raffinose (Raf) synthesis and is involved in plant development and stress responses through regulating Raf content. As a sweetener, Raf makes an important contribution to the sweet taste of white tea. However, studies on the identification, analysis and transcriptional regulation of CsRSs (Camellia sinensis RS genes) are still lacking. In this study, nine CsRSs were identified from the tea plant (Camellia sinensis) genome database. The CsRSs were classified into five groups in the phylogenetic tree. Expression level analysis showed that the CsRSs varied in different parts of the tea plant. Transcriptome data showed that CsRSs could respond to persistent drought and cold acclimation. Except for CsRS5 and CsRS9, the expression pattern of all CsRSs increased at 12 h and decreased at 30 h during the withering process of white tea, consistent with the change trend of the Raf content. Furthermore, combining yeast one-hybrid assays with expression analysis, we found that CsDBB could potentially regulate the expression of CsRS8. Our results provide a new perspective for further research into the characterization of CsRS genes and the formation of the white tea flavour.

Keywords: Camellia sinensis; CsRS8; expression pattern; raffinose synthase; transcriptional regulation; white tea.

Figure 1

The phylogenetic tree, distribution and duplication events of CsRs. (A) Phylogenetic tree of RS proteins constructed using the neighbor-joining(NJ) phylogenetic tree by MEGA X. In total, 57 RS protein sequences from different species were used to construct the phylogenetic tree by MEGA X. The seven different groups are indicated by different colors. Am: Alonsoa meridionalis, Cm: Cucumis melo, Cs: Camellia sinensis, Os: Oryza sativa, Ps: Pisum sativum, Pt: Populus trichocarpa, Rc: Ricinus communis, Si: Sesamum indicum, Va: Vigna angularis, Vv: Vitis vinifera, Zm: Zea mays. The red star represents the RSs in Camellis sinensis. (B) Chromosomal localization and gene duplication events in CsRSs. From outside to inside, the first circle is the chromosome number with the scale on top indicating the coordinate position of the chromosome, and the second circle is the chromosome number and density. Red line indicates gene duplication.

Figure 2

The molecular structure and cis-acting element analyses of CsRSs. (A) Phylogenetic analysis of RS genes in Camellia sinensis. CsRSs are distributed in the three subgroups on the left. (B) Conserved motif of CsRSs. 10 motifs are predicted by MEME and different colored boxes represent different motifs. The box lengths represent motif lengths. (C) Conserved domain structure of CsRSs. The green box represents the RS domain. (D) Exon-intron structures of CsRSs. The green boxes, yellow boxes and black lines represent the UTRs, exons and introns, respectively. (E) Heat map of cis-acting element of CsRSs. Different color blocks represent different promoter types.

Figure 3

The heat map of CsRSs expression mode. (A) Expression pattern of CsRSs in different parts of the tea plant. (B) Expression pattern of CsRSs during continuous drought stress and cold acclimation. The FPKM of the continuous drought stress and cold acclimation converted by log2. PEG-CK: untreated, PEG-24: PEG treatment for 24 h, PEG-48: PEG treatment for 48 h, PEG-72: PEG treatment for 72 h, Cold-CK: nonacclimated at 25~20 °C, CA1-6 h: fully acclimated at 10 °C for 6 h, CA1-7 d: fully acclimated at 10~4 °C for 7 days, DA-7 d: recovering under 25~20 °C for 7 days. (C) Expression pattern of CsRSs in the withering process of white tea.

Figure 4

Raffinose content and CsDBB expression pattern. (A) Changes of Raf content in white tea during withering. (B) Expression pattern of CsDBB in different parts of the tea plant. (C) Expression pattern of CsDBB in the withering process of white tea. Different letters represent significant differences (p < 0.05).

Figure 5

Transcription factor binding site prediction and protein interaction regulation network of CsRSs. (A) Transcription factor binding sites predicted in the promoters of CsRSs. Protein Interaction Regulatory Network of CsRSs. Boxes of different colors represent different transcription factor families. “+” and “−” represent positive and negative strands, respectively. Dotted lines indicate protein interactions, solid lines indicate that transcription factors regulate CsRSs, and the solid red line indicates that it has been verified by experiments. (B) Protein interactions and regulatory networks of CsRSs. The outermost ring represents RS in Camellia sinensis. The circles represent functional genes and the diamonds represent transcription factors, different color blocks represent different families of transcription factors, the dotted line represents the interaction between genes, the solid line represents the regulation of genes by transcription factors, and the red line represents preliminary validation.