Transcriptomic Analysis of Heat Stress Response in Brassica rapa L. ssp. pekinensis with Improved Thermotolerance through Exogenous Glycine Betaine

Abstract

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is sensitive to high temperature, which will cause the B. rapa to remain in a semi-dormancy state. Foliar spray of GB prior to heat stress was proven to enhance B. rapa thermotolerance. In order to understand the molecular mechanisms of GB-primed resistance or adaptation towards heat stress, we investigated the transcriptomes of GB-primed and non-primed heat-sensitive B. rapa 'Beijing No. 3' variety by RNA-Seq analysis. A total of 582 differentially expressed genes (DEGs) were identified from GB-primed plants exposed to heat stress relative to non-primed plants under heat stress and were assigned to 350 gene ontology (GO) pathways and 69 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. The analysis of the KEGG enrichment pathways revealed that the most abundantly up-regulated pathways were protein processing in endoplasmic reticulum (14 genes), followed by plant hormone signal transduction (12 genes), ribosome (8 genes), MAPK signaling pathway (8 genes), homologous recombination (7 genes), nucleotide excision repair metabolism (5 genes), glutathione metabolism (4 genes), and ascorbate and aldarate metabolism (4 genes). The most abundantly down-regulated pathways were plant-pathogen interaction (14 genes), followed by phenylpropanoid biosynthesis (7 genes); arginine and proline metabolism (6 genes); cutin, suberine, and wax biosynthesis (4 genes); and tryptophan metabolism (4 genes). Several calcium sensing/transducing proteins, as well as transcription factors associated with abscisic acid (ABA), salicylic acid (SA), auxin, and cytokinin hormones were either up- or down-regulated in GB-primed B. rapa plants under heat stress. In particular, expression of the genes for antioxidant defense, heat shock response, and DNA damage repair systems were highly increased by GB priming. On the other hand, many of the genes involved in the calcium sensors and cell surface receptors involved in plant innate immunity and the biosynthesis of secondary metabolites were down-regulated in the absence of pathogen elicitors in GB-primed B. rapa seedlings. Overall GB priming activated ABA and SA signaling pathways but deactivated auxin and cytokinin signaling pathways while suppressing the innate immunity in B. rapa seedlings exposed to heat stress. The present study provides a preliminary understanding of the thermotolerance mechanisms in GB-primed plants and is of great importance in developing thermotolerant B. rapa cultivars by using the identified DEGs through genetic modification.

Keywords: B. rapa; HSP; glycine betaine; heat stress; protein processing in endoplasmic reticulum; thermotolerance.

Figure 1

Effects of GB on relative water content (RWC) in B. rapa under heat stress. Data are shown as mean ± SD. Different lowercase letters indicate significant difference at p < 0.05. O-CK, optimum conditions, foliar sprayed with water; Ctrl, foliar sprayed with water before exposure to heat stress; GB, foliar sprayed with GB before exposure to heat stress.

Figure 2

Effects of GB on O₂^·− in B. rapa under heat stress. Data are shown as mean ± SD. Different lowercase letters indicate significant difference at p < 0.05. O-CK, optimum conditions, foliar sprayed with water; Ctrl, foliar sprayed with water before exposure to heat stress; GB, foliar sprayed with GB before exposure to heat stress.

Figure 3

Effects of GB on ATP content in B. rapa under heat stress. Data are shown as mean ± SD. Different lowercase letters indicate significant difference at p < 0.05. O-CK, optimum conditions, foliar sprayed with water; Ctrl, foliar sprayed with water before exposure to heat stress; GB, foliar sprayed with GB before exposure to heat stress.

Figure 4

RNA-Seq data analyses. (A) Heatmap of correlations between samples in different treatments showing three GB and three control (CK) groups. Each value in the heatmap is a Pearson’s correlation coefficient. (B) PCA analysis plot displaying all 6 samples along the axes of PC1 and PC2, which describe 43.7% and 30.1% variability, respectively, within the expression dataset. (C) Volcano plots of the overall DEGs distribution. Threshold was set as an adjusted p-value (Padj) < 0.05 in GB relative to Ctrl. X-axis depicts fold change (FC) in gene expression where the smaller the value, the higher the expression relative to Ctrl. Y-axis depicts false statistical significance of false discovery rate (FDR), where the bigger the value, the smaller the ratio of the number of false positives to the number of total positives. Significantly up-regulated and down-regulated genes are indicated in red and green, respectively. Genes that were not differently expressed are depicted in black. (D) Venn diagram of expressed genes in GB and control under heat stress.

Figure 5

GO enrichment analysis. (A) BP, biology process; (B) CC, cell component; (C) MF, molecular function. The size and color of the point indicates the number of differentially expressed genes in the pathway and the fold change as the threshold for significantly differential expression, respectively.

Figure 6

KEGG pathway enrichment analysis. The vertical axis represents the pathway name, and the horizontal axis represents the gene number ratio of significant DEGs to the total genes in a given KEGG pathway. The size of the dots indicated by “Count” represents the number of DEGs in the pathway, and the color of the dots corresponds to the different q-value ranges. The figure shows the top 18 pathways with q-values. Red and blue letters indicate more than 50% up-regulated and down-regulated DEGs out of a total number of DEGs, respectively.

Figure 7

Gene expression pattern verification by qRT-PCR. A. Three genes for HSP20: BraA03g005240.3.1C, 17.6 kDa class II heat shock protein; BraA01g018430.3.1C, heat shock protein 21 (chloroplastic); BraA01g015830.3.1C, 23.6 kDa heat shock protein (mitochondrial). B. Three genes for antioxidant enzymes: BraA05g010980.3.1C, peroxidase 19; BraA01g023980.3.1C, peroxidase 34; BraA05g031030.3.1C, peroxidase 29 C. Three genes of other pathway genes: BraA08g025430.3.1, glucose-6-phosphate 1-dehydrogenase 3 (chloroplastic); BraA03g050640.3.1C, 5’-adenylylsulfate reductase 3 (chloroplastic); BraA04g029450.3.1C, abscisic acid receptor PYL6.