Transcriptome Profile Analysis of Winter Rapeseed (Brassica napus L.) in Response to Freezing Stress, Reveal Potentially Connected Events to Freezing Stress

Abstract

Winter rapeseed is not only an important oilseed crop, but also a winter cover crop in Northern China, where its production was severely limited by freezing stress. As an overwinter crop, the production is severely limited by freezing stress. Therefore, understanding the physiological and molecular mechanism of winter rapeseed (Brassica napus L.) in freezing stress responses becomes essential for the improvement and development of freezing-tolerant varieties of Brassica napus. In this study, morphological, physiological, ultrastructure and transcriptome changes in the Brassica napus line "2016TS(G)10" (freezing-tolerance line) that was exposed to -2 °C for 0 h, 1 h, 3 h and 24 h were characterized. The results showed that freezing stress caused seedling dehydration, and chloroplast dilation and degradation. The content of malondialdehyde (MDA), proline, soluble protein and soluble sugars were increased, as well as the relative electrolyte leakage (REL) which was significantly increased at frozen 24 h. Subsequently, RNA-seq analysis revealed a total of 98,672 UniGenes that were annotated in Brassica napus and 3905 UniGenes were identified as differentially expressed genes after being exposed to freezing stress. Among these genes, 2312 (59.21%) were up-regulated and 1593 (40.79%) were down-regulated. Most of these DEGs were significantly annotated in the carbohydrates and energy metabolism, signal transduction, amino acid metabolism and translation. Most of the up-regulated DEGs were especially enriched in plant hormone signal transduction, starch and sucrose metabolism pathways. Transcription factor enrichment analysis showed that the AP2/ERF, WRKY and MYB families were also significantly changed. Furthermore, 20 DEGs were selected to validate the transcriptome profiles via quantitative real-time PCR (qRT-PCR). In conclusion, the results provide an overall view of the dynamic changes in physiology and insights into the molecular regulation mechanisms of winter Brassica napus in response to freezing treatment, expanding our understanding on the complex molecular mechanism in plant response to freezing stress.

Keywords: freezing stress; morphology; physiological; transcriptomic; ultrastructure.

Figure 1

The morphology and physiochemical changes of winter type Brassica napus in cold-stress. (A) The seedlings were cold stressed for 24 h in 4, 0, −2, −4 °C and recovered after 24 h. (B) The morphology changes of seedling successive freezing for 24 h. (C) The survival rate, Relative electrolyte leakage, soluble protein, soluble sugar, Proline and MDA content accumulation in leaves of winter Brassica napus stressed with −2 °C for 0, 1, 3 and 24 h. The majuscules indicate a significant difference (p < 0.01) for the data of the stress-treated samples compared with unstressed samples. The mean values were calculated from three biological replicates. Error bars denote standard error of the mean.

Figure 2

The transmission electron micrographs of chloroplast in the mesophyll cell which winter Brassica napus was exposed to in −2 °C with a continuous treatment (0, 1, 3, 24 h). The figure on the right magnification is ×4000; the figure on the right magnification is ×20,000. ch: chloroplast, sg: starch grain, M: mitochondria, CW: cell wall.

Figure 3

The expression profile of chilling regulated differentially expressed genes (DEGs) in Brassica napus leaves. (A) Column diagram representing the numbers of DEGs in three groups. (B) Venn diagrams representing the numbers of DEGs and the overlaps of sets obtained across three comparisons. (C) The heat maps representing 3905 DEGs expression profiles after freezing treatment. (D) the Circos Plot shows that the distribution of DEGs on 19 chromosomes and expression of DEGs in different time points. Red and blue showed the sizes of the 19 chromosomes of B. napus. The purple circle represents the distribution of DEGs on each chromosome. Light blue represents expression (log2 FC) of DEGs at 1 h vs 0 h, light green represents expression (log2 FC) of DEGs at 3 h vs 0 h and yellow represents expression (log2 FC) of DEGs at 24 h vs. 0 h.

Figure 4

Co-expression clustering showing the expression profile of 3905 DEGs. (A) Six major clusters were identified along the time course of cold stress (0, 1, 3 and 24 h). The X-axis represents the time course of freezing stress (0, 1, 3 and 24 h). The Y-axis represents the value of the relative expression level (log2 (FPKM + 1). (B) Functional category enrichment among the six major clusters is based on KEGG annotation.

Figure 5

The functional annotation of differentially expressed genes (DEGs) in Brassica napus leaves under the freezing treatment. (A,B) are GO annotation, (A) up-regulated DEGs, (B) down-regulated DEGs. (C,D) are KEGG pathway enrichment, (C) up-regulated DEGs, (D) down-regulated DEGs.

Figure 6

The distribution of differentially expressed transcription factors in Brassica napus leaves under the freezing treatment. (A) The histograms represent the number of up- or down-regulated transcription factors. (B) The Venn diagrams represent the distribution of transcription factors at different time points.

Figure 7

The qRT-PCR analysis of selected DEG genes in Brassica napus leaves under the freezing treatment. Error bars represent standard errors of the relative expression levels mean values by qRT-PCR (n = 4) (left y-axis). Broken lines represent transcript levels change (log2 FC) according to the FPKM value of RNA-Seq (right y-axis). Correlation between qRT-PCR and RNA-seq for select DEGs is also shown.

Figure 8

The differential responses of Brassica napus in response to freezing stress in transcriptome changes.