Transcriptomic Response of Chinese Yew (Taxus chinensis) to Cold Stress

Abstract

Taxus chinensis is a rare and endangered shrub, highly sensitive to temperature changes and widely known for its potential in cancer treatment. How gene expression of T. chinensis responds to low temperature is still unknown. To investigate cold response of the genus Taxus, we obtained the transcriptome profiles of T. chinensis grown under normal and low temperature (cold stress, 0°C) conditions using Illumina Miseq sequencing. A transcriptome including 83,963 transcripts and 62,654 genes were assembled from 4.16 Gb of reads data. Comparative transcriptomic analysis identified 2,025 differently expressed (DE) isoforms at p < 0.05, of which 1,437 were up-regulated by cold stress and 588 were down-regulated. Annotation of DE isoforms indicated that transcription factors (TFs) in the MAPK signaling pathway and TF families of NAC, WRKY, bZIP, MYB, and ERF were transcriptionally activated. This might have been caused by the accumulation of secondary messengers, such as reactive oxygen species (ROS) and Ca²⁺. While accumulation of ROS will have caused damages to cells, our results indicated that to adapt to low temperatures T. chinensis employed a series of mechanisms to minimize these damages. The mechanisms included: (i) cold-enhanced expression of ROS deoxidant systems, such as peroxidase and phospholipid hydroperoxide glutathione peroxidase, to remove ROS. This was further confirmed by analyses showing increased activity of POD, SOD, and CAT under cold stress. (ii) Activation of starch and sucrose metabolism, thiamine metabolism, and purine metabolism by cold-stress to produce metabolites which either protect cell organelles or lower the ROS content in cells. These processes are regulated by ROS signaling, as the "feedback" toward ROS accumulation.

Keywords: DE isoforms; ROS; Taxus chinensis; cold response; cold stress; transcriptome.

Figure 1

Enzyme activity of CAT, POD, and CAT in leaf tissues of Taxus chinensis grown under normal and cold stress conditions. (A) Hydroperoxidase (CAT), (B) Peroxidase (POD), and (C) Superoxide Dismutase (SOD). Leaf tissues were sampled from plants grown under normal conditions and from plants that were subjected to 0°C for 1, 2, 4, 8, 12, 24, and 48 h. Results are means and SD for three replicates.

Figure 2

Differently expressed transcripts between control and cold stressed samples. (A) MA plot, (B) Volcano plot, and (C) Number of transcripts that expressed most differently at different significant levels. The red dots in MA and volcano plots indicated the transcripts that were differently expressed at least a fold change ratio of four (|log₂FC| > 2) between control and cold stressed samples.

Figure 3

Classification of transcription factors (TFs) that were significant (p < 0.05 and |log2FC| > 2) changed under cold stressed conditions. Others in cold up regulated include: AP2(1), ARR-B(1) B3(1), BES1(1), Dof(1), E2F/DP(1), G2-like(1) GATA HSF(1), LBD(1), M-type MADS(1), NF-YA(1), NF-YC(1), Nin-like(1), Trihelix(1), CO-like(1), ARF(2), and TALE(2); Others in cold: DBB(1), NF-YB(1), SBP(1), and CO-like(1). Up, cold up regulated; Down, cold down regulated.

Figure 4

KEGG path way annotation for transcripts that were significantly (p < 0.05) affected by cold stress and had at least a fold-change of four (|log₂FC|> 2). (A) Overall classification and (B) sub-classification of metabolic pathways. Up, cold up-regulated transcripts; Down, cold down-regulated transcripts.

Figure 5

Networks of GO terms enriched in DE ORFs at p < 0.05 level and had at least a 4-folderexpression ratio change (log₂FC > 2) in response to cold stress. (A) GO terms enriched from cold up regulated ORFs, (B) GO terms enrichd from cold down regulated ORFs. GO enrichment analysis were carried out using BinGo after ORFs annotated to Arabidopsis. The lines between GO terms indicated their parent-child relationships. Sizes of circles represent the size of GO terms in Arabidopsis annotation. Colors indicate p-values of Benjamini and Hochberg False Discovery Rate (FDR) correction. Detailed information of GO terms are list in Table 1.