Transcriptome and Small-RNA Sequencing Reveals the Response Mechanism of Brassica napus to Waterlogging Stress

Abstract

Rapeseed (Brassica napus) is highly susceptible to waterlogging during the seedling stage; however, most of the studies on its gene expression under waterlogging stress have focused on transcriptional regulation, with little work conducted on post-transcriptional regulation to date. To elucidate this regulatory network, comparative transcriptome and miRNA analyses in the leaves and roots of rapeseed Zhongshuang11 (ZS11) were performed. Differentially expressed genes (DEGs) and miRNAs (DEmiRNAs) were identified by comparing the normal planting condition (the control group, CKT) with waterlogging treatment (WLT). DEGs identified in leaves and roots were enriched in different metabolic pathways, indicating their distinct mechanisms in response to waterlogging stress. In total, 68 and 82 DEmiRNAs were identified in leaves and roots, respectively, predicted to target 543 and 2122 DEGs in each tissue. Among these, 12 and 9 transcription factors (TFs) were exclusively targeted by DEmiRNAs in leaves and roots, respectively. Notably, six upregulated TFs in leaves were associated with the ethylene response and were predicted targets of bna-miR172 family members, and four TFs in roots participated in the ethylene response pathway. Furthermore, bna-miR169, along with novel-miR-23108 and novel-miR-42624 family members, played crucial roles in waterlogging response of rapeseed. Combining with the determination results of ethylene and jasmonic acid content, a preliminary model of miRNA-mediated gene expression regulation in rapeseed response to waterlogging stress was developed. These findings advance our understanding of transcriptional regulation under waterlogging and lay a theoretical foundation for improving rapeseed waterlogging tolerance.

Keywords: Brassica napus; comparative transcriptome; microRNA; waterlogging.

Figure 1

Morphological and physiological changes in rapeseed under waterlogging. (A) Morphological responses of ZS11 to waterlogging stress. Scale bar = 4 cm. (B) Waterlogging-induced reduction in leaf net photosynthetic rate and stomatal conductance. (C) Decline in leaf chlorophyll content and SPAD values under waterlogging. (D) Decreased aboveground biomass (fresh and dry weight) under waterlogging. (E) Reduced underground biomass (fresh and dry weight) due to waterlogging. (F) Accumulation of leaf MDA content under waterlogging stress. (G) Increased soluble sugar content in leaves under waterlogging. (H) Jasmonic acid accumulation in leaves and roots after 8 days of waterlogging. (I) ACC content (ethylene precursor) in leaves and roots after 8 days of waterlogging. Note: In the figure, “ns” indicates no significant difference, “*” represents significant differences from the CK at the same time point (p < 0.05), “**” indicates extremely significant differences (p < 0.01), “***” indicates p < 0.001, and “****” indicates p < 0.0001. CK: control; WL: waterlogging.

Figure 2

GO enrichment analysis of DEGs identified in roots and leaves. (A) GO enrichment analysis of common DEGs in both leaves and roots. (B) GO enrichment analysis of leave-specific DEGs. (C) GO enrichment analysis of root-specific DEGs.

Figure 3

KEGG pathway classification of DEGs identified in roots and leaves. (A) KEGG pathway of common DEGs in both leaves and roots. (B) KEGG pathways of leave-specific DEGs. (C) KEGG pathways of root-specific DEGs.

Figure 4

Base length distribution of miRNAs and miRNA families in leaves and roots. (A) miRNAs length distribution in roots. (B) miRNAs length distribution in leaves. (C) Length profiles of known miRNA families in leaves. (D) Length profiles of novel miRNA families in leaves. (E) Length profiles of known miRNA families in roots. (F) Length profiles of novel predicted miRNA families in roots.

Figure 5

GO enrichment analysis of DEGs targeted by DEmiRNAs. (A) GO enrichment analysis of DEGs targeted by DEmiRNAs in leaves. (B) GO enrichment analysis of DEGs targeted by DEmiRNAs in roots. (C) GO enrichment analysis of DEGs targeted by novel-miR-23108 in leaves. (D) GO enrichment analysis of DEGs targeted by novel-miR-42624 in roots.

Figure 6

Expression analysis of the 21 TFs and the 2 novel miRNAs. (A) Transcriptome-based expression profiles of the 21 TFs and the 2 novel miRNAs. (B) RT-qPCR validation of TF expression levels, stem-loop RT-qPCR validation of miRNA expression levels. Note: In the figure, the nine TFs listed above the red line were identified in roots, while the twelve TFs below the red line were identified in leaves.

Figure 7

Schematic diagram of gene expression regulation in rapeseed under waterlogging stress.