Global transcriptome profiles of Camellia sinensis during cold acclimation

Abstract

Background: Tea is the most popular non-alcoholic health beverage in the world. The tea plant (Camellia sinensis (L.) O. Kuntze) needs to undergo a cold acclimation process to enhance its freezing tolerance in winter. Changes that occur at the molecular level in response to low temperatures are poorly understood in tea plants. To elucidate the molecular mechanisms of cold acclimation, we employed RNA-Seq and digital gene expression (DGE) technologies to the study of genome-wide expression profiles during cold acclimation in tea plants.

Results: Using the Illumina sequencing platform, we obtained approximately 57.35 million RNA-Seq reads. These reads were assembled into 216,831 transcripts, with an average length of 356 bp and an N50 of 529 bp. In total, 1,770 differentially expressed transcripts were identified, of which 1,168 were up-regulated and 602 down-regulated. These include a group of cold sensor or signal transduction genes, cold-responsive transcription factor genes, plasma membrane stabilization related genes, osmosensing-responsive genes, and detoxification enzyme genes. DGE and quantitative RT-PCR analysis further confirmed the results from RNA-Seq analysis. Pathway analysis indicated that the "carbohydrate metabolism pathway" and the "calcium signaling pathway" might play a vital role in tea plants' responses to cold stress.

Conclusions: Our study presents a global survey of transcriptome profiles of tea plants in response to low, non-freezing temperatures and yields insights into the molecular mechanisms of tea plants during the cold acclimation process. It could also serve as a valuable resource for relevant research on cold-tolerance and help to explore the cold-related genes in improving the understanding of low-temperature tolerance and plant-environment interactions.

Figure 1

Changes of relative electrical conductivity and of the averaged air temperature during the CA process of tea plants. The value of relative electrical conductivity changed from 96.2% for sample CK to 42.9% for sample CA1 when tea plant underwent the cold acclimation process. Then, it returned back to 80.3% (sample CA3) after the air temperature increased. The values of averaged air temperature were also shown during this period.

Figure 2

Venn diagram showing the BLAST results of C. sinensis transcriptome against five databases. De novo reconstructed transcript sequences were used to BLAST search against public databases including NCBI’s NR, UniRef90, TAIR10, KEGG and KOG. The number of transcripts that have significant hits (E-value ≤ 10^–5) against the five databases is shown in each intersection of the Venn diagram.

Figure 3

Protein families in C. sinensis transcriptome. Shown are the number of Pfam domains/families versus the occurrence of C. sinensis transcripts contained in each domain/family (A), and the 10 most abundant protein families (B) in C. sinensis.

Figure 4

Heatmap representing relative expression levels of 398 differentially expressed transcripts in tea plants. Expression levels were determined by RNA-Seq and DGE, respectively. Cluster analysis of differentially regulated transcripts expressed in CA1 versus CA and CA3 samples indicating the high degree of consistency between DGE and RNA-Seq platforms.

Figure 5

Quantitative RT-PCR validations. 18 genes were selected for the quantitative RT-PCR analysis, including catalase (A), thaumatin-like protein (B), ascorbate peroxidase (C), trehalose-6-phosphate synthase (D), bZIP (E), early-responsive to dehydration stress protein (F), phospholipase (G), absicisic acid 8′-hydroxylase (H), HSP70 (I), E3 ubiquitin-protein ligase (J), WRKY2 (K), sensor histidine kinase (L), MYB (M), peroxidase (N), delta-6-desaturase (O), Aquaporin 2 (P), ABA receptor (Q) and bHLH (R). The18S ribosomal gene was chosen as the reference gene.

Figure 6

A diagram of tea plants’ responses to low temperatures during the CA process.