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Identifying Differentially Expressed Genes Associated with Tolerance against Low Temperature Stress in Brassica napus through Transcriptome Analysis.

Byline: Mengzhu Xian, Tao Luo, Mohammad Nauman Khan, Liyong Hu and Zhenghua Xu


Under direct-seeding method of sowing, seedling survival is adversely affected due to low temperature. The purpose of this study was to understand the morphological, physiological and molecular response of rapeseed resistance to low temperature stress at seedling establishment. Two varieties Qinyou No.7 (resistant biotype) and Fengyou 730 (sensitive biotype) were used for morphological and physiological experiments under low temperature stress and the resistant variety was used for transcriptome analysis based on the Illumina HiSeq 2000 platform. Results showed resistant variety initiated osmotic regulation system firstly and then triggered the antioxidant enzyme system when exposed to low temperature stress for long duration. The MDA content firstly tended to increase and then decreased in resistant variety, but it continued to increase in sensitive variety.

Through transcriptome analysis of Qinyou No. 7 under low temperature for 0 h, 24 h, 48 h and 96 h, 2254 differentially expressed unigenes clustering in 7 expression patterns were participated in low temperature stress. Protein-serine/threonine kinases, myo-inositol-1-phosphate synthases and calmodulins, as the members of ABA and IP3/Ca2+ signal transduction pathway, played an important role in the low temperature stress. Different expressed genes dataset provides useful candidate genes for functional analysis of rapeseed resistance to low temperature.

Keywords: Brassica; Low temperature stress; Transcriptome; Seedling growth


Rapeseed (Brassica napus L.) is one of the most important oilseed crops in the world because it is edible oil source for human and a potential future source for renewable biofuel (Tian et al., 2016). The traditional method of seedling transplantation, which demands more labor force and cost, is nowadays replaced with direct-seeding method (Wang et al., 2015). Healthy seedling establishment is a crucial developmental stage to determine the population density of rapeseed (Brassica napus L.) in field. However, reduction in seedling survival due to low temperature under direct-seeding is the limitation for the high yield acquisition and the promotion of planting area.

Previous researches revealed that the seasonal cold climate had become a direct obstacle for seedling establishment (Zheng et al., 1998; Diepenbrock, 2000). An analysis of morphological traits under low temperature conditions indicated that seedling height, and shoot dry weights decreased as temperature decreased in sorghum (Yu et al., 2004). Higher concentrations of soluble sugars, glucose and proline were positively associated with low temperature stress, which acted as osmotic substances to preserve protein structure and function (Patton et al., 2007; Burbulis et al., 2011; Trischuk et al., 2014; Farooq et al., 2016). Low temperature stress induced the accumulation of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radicals (Suzuki and Mittler, 2006; Farooq et al., 2009).

These ROS may be signals inducing ROS scavengers and other protective mechanisms, as well as damaging agents contributing to stress injury in plants (Xiong et al., 2002; Airaki et al., 2012; Li et al., 2013; ; Farooq et al., 2016)). Diverse plant species tolerate cold stress to a varying degree, which depends on reprogramming gene expression to modify their physiology, metabolism, and growth (Chinnusamy et al., 2010). With the rapid advance of next-generation sequencing (NGS) technology, a large number of low temperature regulated genes had been identified to involve in a variety of biological processes in many crops (Xu et al., 2011; Secco et al., 2013; Zhang et al., 2013; An et al., 2015; Huang et al., 2015). Complex gene networks, especially the unsaturated fatty acid and jasmonic acid biosynthesis pathways, were involved in the cold stress in C. Japonica (Li et al., 2016).

Many heat shock protein genes were triggered in plant cells when exposed to low temperature stress in Seabuckthorn (Sharma and Chaudhary, 2016). Information on the low-temperature transcriptome, proteome and metabolome is expected to continue to increase in the near future. This information is necessary for our understanding of the complex network of molecular changes that are important for low temperature tolerance (Zhu et al., 2007). There are rare transcriptomic studies for seedling establishment of rapeseed to identify and characterize their cold-stress-induced gene expression.

To our knowledge, a comprehensive research from morphological physiological and molecular levels to study the effect of low temperature on the seedling establishment of rapeseed (Brassica napus L.) is still rare. The present study was to explore the low temperature resistance mechanisms in resistant and susceptible biotypes of rapeseed by morphological and physiological measurement and transcriptome analysis at the seedling establishment stage. The objectives of this study were to reveal the different morphological and physiological responses of resistant and sensitive varieties to low temperature stress, and to identify different expressed genes participated in low temperature stress, further to provide useful candidate genes for functional analysis and successful seedling establishment of rapeseed resistance to low temperature.

Materials and Methods

Plant Materials and Growth Conditions

Two varieties of Qinyou No. 7 (resistant biotype) and Fengyou 730 (sensitive biotype) were used for morphological and physiological experiments (Xian et al., 2015). While the resistant variety Qinyou No. 7 was used for transcriptome analysis. Newly-harvested seeds of Qinyou No. 7 and Fengyou 730 came from The Research Center of Rapeseed of Shanxi province and The Crops Research Institute of Hunan Province, China. For each variety, one hundred seeds (arranged in 10 x 10) were sown in each germination box (12 cmx12 cmx6 cm) and covered with 3-layer sterilized filter paper. The filter paper was moistened with 10 mL Hoagland's solution and 1 mL solution was added every day during the experimental period to provide adequate water for germination and seedling establishment. Seeds were germinated in incubator (day/night temperature at 25/20degC) with a photoperiod of 12 h light (150 umol photons m-2 s-1) and 12 h dark at 50% relative humidity.

After 3 days, half of the germination boxes for each variety were placed in incubator (day/night temperature at 15/10degC) for low temperature treatment; the other germination boxes were placed in incubator (day/night temperature at 25/20degC) for normal temperature treatment.

Roots and shoots for the low temperature treatment and the control were harvested separately at the time points of 0 h, 24 h, 48 h and 96 h after the low temperature treatment began.

Morphological and Physiological Parameters Measurement

Seedling morphological parameters such as shoot length (SL), root length (RL), total length (TL), shoot fresh weight (SFW), root fresh weight (RFW), total fresh weight (TFW), total dry weight (TDW), ratio of root to shoot length (RL/SL), ratio of root to shoot weight (RFW/SFW), ratio of shoot dry to fresh weight (SDW/SFW), ratio of root dry to fresh weight (RDW/RFW), and ratio of total dry to fresh weight (TDW/TFW) were recorded at each sampling time point. The harvested materials were dried to a constant weight at 80degC to measure the shoot dry weight (SDW) and root dry weight (RDW).

Shoot material was collected for the determination of physiological parameters such as the content of proline, MDA and soluble protein, and the enzyme activity of SOD, POD and CAT. Proline was extracted with 5-sulfosalicylic acid, quantified by means of a colorimetric reaction with ninhydrin according to the method of Li Heseng (Li, 2000). MDA content was estimated by shoot homogenates reacted with thiobarbituric acid according to the method of Bhatugar-Mathur (Bhatnagar-Mathur et al., 2009). Soluble protein content was determined by Coomassie brilliant blue (G-250) according to the Bradford method (Bradford, 1976). SOD activity was determined by measuring its ability to inhibit photochemical reduction of nitroblue tetrazolium (NBT) as described by Donahue (Donahue et al., 1997).

POD activity was determined by measuring its ability to catalyze the colorimetric reaction of hydrogen peroxide (H2O2) and guaiacol as described by Raza et al. (2007). CAT activity was determined by measuring its ability to degrade the concentration of H2O2 as described by Raza et al.(2007).

RNA Extraction, Library Preparation and Sequencing

The four RNA samples, including 3 samples from the rapeseed shoots after 24 h, 48 h and 96 h chilling treatment and a mixed sample of shoots were harvested under control condition (25/20degC) at the same sampling time point, were collected from three replications, the replications for each treatment were pooled together to make one sample for transcriptome analysis.

Total RNA was extracted using TRK-1001 total RNA purification kit (LC Science, Houston, TX) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0.

Approximately 5 ug of total RNA representing a specific adipose type was subjected to isolate poly (A) mRNA with poly-T oligo attached magnetic beads (thermo-fisher). Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the d UTP method as described, the average insert size for the paired-end libraries was 300 (+-50 bp). And then we performed the paired-end sequencing on an Illumina Hiseq 2000 (LC Sceiences, USA) platform using 100 bp paired-end reads.

Transcriptome Data Processing

All RNA-seq datas were aligned to Brassica napus L. reference genome (build 5) using Top Hat v2.0.9 with default parameters. The mapped reads were assembled using Cufflinks v2.11. All multiple assembled transcript files were then merged to produce a unique set of transcriptomes using the Cuffmerge utility provided by the Cufflinks package. Cuffdiff v2.11 was used for all differential expression analyses. Transcript abundances were estimated by Cufflinks in Fragments per Kilobase per Million mapped reads (FPKM) for paired-end reads. The differentially expressed genes (24 h compared with 0 h, 48 h compared with 0 h and 96 h compared with 0 h) were selected using a threshold of absolute value of log2 (fold change) >1 and FDR 99%) were used for further analysis. The raw sequence data have been submitted to the NCBI Short Read Archive with accession number SRP067875. More than 77% of sample reads matched to the reference database (build 5), and more than 90% of these matching reads were positioned into exons region of the reference genome (see additional file 2 and 3 ).

Table 1: Throughput and quality of RNA-seq for the 4 libraries from low temperature treatment of 0 h, 24 h, 48 h and 96 h

Library###Raw Data###Valid Data###Valid Ratio (%)###Q20%

###Reads###Base (G)###Reads###Base (G)


LT 24h###29,258,470###3.66###28,806,142###3.6###98.45###99.38

LT 48h###35,524,816###4.44###35,033,202###4.38###98.62###99.38

LT 96h###32,984,540###4.12###32,448,932###4.06###98.38###99.25

There were 101040 genes assembled using the valid reads, in which 52086 genes showed significant annotated in GO database and 21218 genes were mapped to 269 KEGG pathways.

Differential Expression Gene Analyses

Compared with the control, a total number of 2001, 2001 and 1998 differentially expressed genes (DEGs) were discovered under low temperature treatment of 24 h, 48 h and 96 h, respectively. The number of up-regulated genes was increased by 40 when the low temperature treatment continued from 0 to 96 h. There were 428 up-regulated genes and 381 down-regulated genes among the LT-24 h vs. CK, LT-48h vs. CK and LT-96h vs. CK (Fig. 4).

The distribution of these genes is shown in Figure 5. Under biological processes, metabolic process was the most abundant GO term (13.6%), followed by cellular process (11.3%) and single-organism process (10.2%). In molecular function, genes-encoding proteins responsible for binding (18.2%) and catalytic activity (12.4%) were highly represented. For cellular component, the major categories included cell (4.9%), cell part (4.9%) and organelle (2.8%).

The KEGG pathway analysis showed that the most abundant pathways were related to metabolism (777 unigenes). Of the metabolic pathways that were identified, the most likely linked to low temperature stress is carbohydrate metabolism (206), followed by amino acid metabolism (158). There are 50 unigenes participated in low temperature-induced signal transduction (see additional file 4). Auxin responsive and transport protein, abscisic acid receptor, serine/threonine protein kinases and calmodulin were involved in signal transduction under low temperature stress.

Profiling Responses to the Low Temperature Duration

Using STEM clustering method to define a set of distinct and representative temporal gene expression mode under the four low temperature duration treatments, 2254 unigenes were clustered into 7 profiles (Fig. 7, including 3 up-regulated patterns (1248 unigenes) and 4 down-regulated patterns (1006 unigenes).

The continued up-regulated (profile 19) and down regulated (profile 0) DEGs were subjected to KEGG pathway analysis (Fig. 8). The KEGG enrichment analysis showed that the unigenes mapped to protein processing in endoplasmic reticulum and protein export were up-regulated with the duration of low temperature ranging from 0-96 h, which demonstrated that the Qinyou No. 7 significantly increased protein synthesis and processing with positive transcriptional regulation to resist low temperature. Transcriptions related to the starch and sucrose metabolism were also increased.

Validation of the Different Expressed Genes by qRT-PCR

To confirm the accuracy and reproducibility of the transcriptome analysis results, 12 unigenes (8 from continue to the up-regulated unigenes profile and 4 from continue to down-regulated unigenes profile) were selected for real-time quantitative reverse transcription PCR (qRT-PCR) validation. The expression profiles of the candidate unigenes revealed by qRT-PCR data were consistent with those derived from sequencing (Fig. 9).


As the intuitionistic behavior of rapeseed seedling response to low temperature stress, morphological characteristics showed that seedling establishment was significantly inhibited under low temperature stress. Alteration in root-shoot length ratio was observed in this study and such kind of results were obtained for wheat crop (Equiza et al., 2001). It was interesting that the dry weight of shoot was higher under low temperature than under normal temperature, which probably was associated with respiration inhibition under low temperature (Hurry et al., 1995). These findings are supported by the previous study on rapeseed (Ping et al., 2015), but are inconsistent with the results reported about sorghum (Yu et al., 2004). Transcriptome analysis showed that the expression of many genes related to indole-3-acetic acid synthetase and auxin responsive and transport protein were down-regulated under low temperature stress.

Indole-3-acetic acid (IAA), the main auxin in higher plants, has profound effects on plant growth and development (Zhao, 2010; Mashiguchia et al., 2011). This implied that the auxin metabolism participated in controlling the length of root and shoot under low temperature stress at seedling establishment stage.

Changes in physiological characteristics reflected the process of seedling establishment responding to low temperature stress and the injury degree of stress. Plants under low temperature stress increased soluble matter concentration of cell to enhance the osmotic potential and drop the freezing point (Stitt and Hurry, 2002; Zhu et al., 2007). In this study, the concentration of proline and soluble sugar were increased in shoots under low temperature, and the increase in resistant variety was significantly higher than sensitive variety. Similar results were also found in previous researches (Kushad and Yelenosky, 1987; Patton et al., 2007; Burbulis et al., 2011; Li et al., 2013). Carbohydrate and protein metabolism is the foundation for the rapeseed seedling establishment (Patton et al., 2007; Lutz, 2010).

Transcriptome results revealed that beta-amylase and beta-glucosidase genes expression were up-regulated in low temperature stress to increase the sugar content. There are 8 heat shock protein expression, in which the heat shock 70kDa protein related gene BnaA03g14210D and BnaA03g17100D continued to up-regulate during the low temperature stress. The expression of molecular chaperone gene BnaC05g35680D was up-regulated firstly from 0-24 h and then down-regulated from 24-96 h in low temperature treatment. The up-regulated expression of molecular chaperone and heat shock protein helped to maintain the correct assembly of peptide and structure stability of protein, and then enhanced the resistance to low temperature (Hartl et al., 2011; Aghdam et al., 2013).

Under low temperature stress, the reactive oxygen species (ROS) levels were raised in plant cells and the lipid peroxidation of the cell membrane was intense, which resulted in the accumulation of some toxic metabolites in plant (Wise and Naylor, 1987). As a product of membrane lipid peroxidation, MDA content in tissue reflected the degree of stress damage of biological membrane (An et al., 2012). The MDA content firstly tended to increase and then decreased in resistant variety, but it continued to increase in sensitive variety. Research about physiological response of rapeseed to chilling stress showed that resistance materials persisted less MDA content under chilling stress (Pu and Sun, 2010; Zhang et al., 2015b). Peroxidases are a kind of antioxidant enzymes, which can remove ROS in plant cell (Breusegem et al., 1999).

Previous studies mainly focused on SOD, POD and CAT activities, however, due to the material properties and the difference of stress duration and intensity, changes in antioxidant enzymes under low temperature stress were not so regular (Gechev et al., 2003; Zhang et al., 2011; Li et al., 2013). In our experiment, the resistant variety initiated osmotic regulation system firstly and then triggered the antioxidant enzyme system when exposed to low temperature stress for long duration. The enzyme activity of sensitive material increased slightly earlier than resistant variety, but along with extension of low temperature stress, CAT activity of sensitive variety gradually reduced. Expression of 5 peroxidases genes increased in resistant variety, among which the BnaA06g16150D expression was up-regulated to 5 times of the control as the low temperature duration extended from 0-96 h in resistant variety.

There were 12 glutathione S-transferase (GST) genes up-regulated. Overexpression of GST can stimulate seedling growth under chilling and salt stress, and this effect could be caused by oxidation of the glutathione pool (Roxas et al., 1997). Overexpression of a tobacco glutathione S-transferase with glutathione peroxidase activity (GST/GPX) in transgenic tobacco (Nicotiana tabacum L.) enhanced seedling growth under a variety of stressful conditions (Roxas et al., 2000). Secondary metabolism and its products also play an important role in rapeseed response to low temperature stress response. There were 8 flavonol synthase genes up-regulated, in which BnaC07g29940D expression continued to rise. The flavonoids from phenylpropanoid pathway could also protect membrane lipid in cold stress (Crifo et al., 2011; Gao et al., 2013).

The mechanism of low temperature response in rapeseed seedling at establishment stage is very complex, including the chilling signal transduction induced related genes expression and its metabolites involved in this process. A generic signal transduction pathway starts with the low temperature stress, followed by the generation of second messengers (Shinozaki et al., 2003; Chinnusamy et al., 2007). Second messengers can modulate intracellular Ca2+ levels, often initiating a protein phosphorylation cascade that finally targets proteins directly involved in cellular protection or transcription factors controlling specific sets of stress-regulated genes (Xiong et al., 2002; Kudla et al., 2010; Batistic and Kudla, 2012). IP3 levels increased in plants under stress, and the time frame for the increase correlated with changes in cytosolic Ca2+ levels (Dewald et al., 2001).

In our experiment, 4 myo-inositol-1-phosphate synthase genes and 3 calmodulin genes participated in the resistance to low temperature stress, and the expression of calmodulin genes continued to increase during low temperature treatment. It demonstrates that IP3/Ca2+ signal transduction pathway plays an important role in the low temperature stress resistance. Many transcription factors have been reported in response to cold stress and alter the expression levels of some cold stress-responsive genes, eventually affect several physiological traits to overcome the adverse conditions (Gilmour et al., 1998; Kasuga et al., 1999; Sakuma et al., 2002). Fourteen transcription factors from zinc finger (2), MYB (9), MADS (3) were considered to be associated with low temperature stress in seedling establishment.

Among these, the expression of the zinc finger, 7 of the MYB and 1 of the MADS were up-regulated. Protein-serine/threonine kinases are one of the key kinases in ABA signal transduction pathway, regulating the activity of some protein by the phosphorylation (Mizoguchi et al., 1995; Brock et al., 2010; Roskoski, 2010). The up-regulated expression of the protein-serine/threonine kinases in low temperature stress reveals that ABA signal transduction pathway participates in the low temperature stress at seedling establishment stage.


Complex mechanisms including morphological, physiological and transcriptional level were involved in seedling establishment of rapeseed responding to low temperature stress. Shoot and root elongation was inhibited under low temperature stress, but the dry weight was increased. The resistant variety firstly initiated osmotic regulation system, and then triggered the antioxidant enzyme system under low temperature stress. Through transcriptome analysis, 2254 differentially expressed unigenes clustering in 7 expression patterns were participated in resistance to low temperature stress. The differentially expressed genes dataset will also be served as a very useful genetic resource to analysis the resistance of rapeseed to low temperature. Further studies are needed to verify the functions of candidate genes for improving low temperature tolerance ability through genetic engineering.


This research was funded by National Natural Science Foundation of China (31301268), Project 2013QC043 supported by the Fundamental Research Funds for the Central Universities and National Modern Rapeseed Industry Technology System (NYCYTC-00510).


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Author:Xian, Mengzhu; Luo, Tao; Khan, Mohammad Nauman; Hu, Liyong; Xu, Zhenghua
Publication:International Journal of Agriculture and Biology
Article Type:Report
Date:Apr 30, 2017
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