Transcriptome profiling reveals candidate flavonol-related genes of Tetrastigma hemsleyanum under cold stress

Abstract

Background: Tetrastigma hemsleyanum Diels et Gilg is a valuable medicinal herb, whose main bioactive constituents are flavonoids. Chilling sensitivity is the dominant environmental factor limiting growth and development of the plants. But the mechanisms of cold sensitivity in this plant are still unclear. Also, not enough information on genes involved in flavonoid biosynthesis in T. hemsleyanum is available to understand the mechanisms of its physiological and pharmaceutical effects.

Results: The electrolyte leakage, POD activity, soluble protein, and MDA content showed a linear sustained increase under cold stress. The critical period of cold damage in T. hemsleyanum was from 12 h to 48 h. Expression profiles revealed 18,104 differentially expressed genes (DEGs) among these critical time points. Most of the cold regulated DEGs were early-response genes. A total of 114 unigenes were assigned to the flavonoid biosynthetic pathway. Fourteen genes most likely to encode flavonoid biosynthetic enzymes were identified. Flavonols of T. hemsleyanum might play a crucial role in combating cold stress. Genes encoding PAL, 4CL, CHS, ANR, FLS, and LAR were significantly up-regulated by cold stress, which could result in a significant increase in crucial flavonols (catechin, epicatechin, rutin, and quercetin) in T. hemsleyanum.

Conclusions: Overall, our results show that the expression of genes related to flavonol biosynthesis as well as flavonol content increased in T. hemsleyanum under cold stress. These findings provide valuable information regarding the transcriptome changes in response to cold stress and give a clue for identifying candidate genes as promising targets that could be used for improving cold tolerance via molecular breeding. The study also provides candidate genes involved in flavonoid biosynthesis and may be useful for clarifying the biosynthetic pathway of flavonoids in T. hemsleyanum.

Keywords: Cold stress; Flavonol; Tetrastigma hemsleyanum; Transcriptome.

Fig. 1

Physiological changes of T. hemsleyanum leaves under 25 °C (a) or 0 °C (b) exposure across seven time points (0, 4, 8, 12, 24, 48, 72 h)

Fig. 2

Length distribution of transcripts in T. hemsleyanum transcriptome

Fig. 3

Gene ontology classification of the T. hemsleyanum transcriptome

Fig. 4

COG functional classification of the T. hemsleyanum transcriptome

Fig. 5

Transcriptional profiles of T. hemsleyanum after 0 to 48 h of cold exposure. a Distribution of genes differentially expressed under 0 °C exposure for 12 h, 24 h, and 48 h of cold exposure, b Hierarchical clustering analysis of the commonly expressed DEGs from the RNA-Seq in each treatment (yellow, induced genes; blue, repressed genes)

Fig. 6

Heatmap of the overall changes of flavonoid metabolism-related genes in T. hemsleyanum after 0 to 48 h of cold exposure

Fig. 7

The schematic diagram of the flavonoid biosynthetic pathways. The heatmap presents the expression levels of flavonoid biosynthetic genes based on T. hemsleyanum RNA sequencing data. The first number in brackets following each gene name refers to the number of transcriptome unigenes annotated as that gene, while the red and green numbers on the arrows indicated the number of significantly up-regulated and down-regulated unigenes, respectively. A color bar is presented at the bottom right. Data represent the log2 values (RPKM) of the T. hemsleyanum during the CK, Z12h, Z24h, and Z48h stages (from left to right)

Fig. 8

The expression of favonol-related genes of T. hemsleyanum after 0 to 48 h of cold exposure. a The genes expression as determined by qRT-PCR. b Comparison between the log2 of gene expression ratios obtained from RNA-seq data and qRT-PCR. The qPCR log2value of the expression ratio (cold-acclimated/CK) (y-axis) was plotted against the value from the RNA-seq (x-axis). All qPCR data were collected from three biological replicates and three technical replicates for each sample

Fig. 9

The HPLC chromatograms of reference standards and T. hemsleyanum sample. 1 procyanidins B₁; 2. catechin; 3. procyanidinsB₂; 4. epicatechin; 5. rutin; 6. quercetin; 7. kaempferol