Integrated transcriptome and small RNA sequencing analyses reveal a drought stress response network in Sophora tonkinensis

Abstract

Background: Sophora tonkinensis Gagnep is a traditional Chinese medical plant that is mainly cultivated in southern China. Drought stress is one of the major abiotic stresses that negatively impacts S. tonkinensis growth. However, the molecular mechanisms governing the responses to drought stress in S. tonkinensis at the transcriptional and posttranscriptional levels are not well understood.

Results: To identify genes and miRNAs involved in drought stress responses in S. tonkinensis, both mRNA and small RNA sequencing was performed in root samples under control, mild drought, and severe drought conditions. mRNA sequencing revealed 66,476 unigenes, and the differentially expressed unigenes (DEGs) were associated with several key pathways, including phenylpropanoid biosynthesis, sugar metabolism, and quinolizidine alkaloid biosynthesis pathways. A total of 10 and 30 transcription factors (TFs) were identified among the DEGs under mild and severe drought stress, respectively. Moreover, small RNA sequencing revealed a total of 368 miRNAs, including 255 known miRNAs and 113 novel miRNAs. The differentially expressed miRNAs and their target genes were involved in the regulation of plant hormone signal transduction, the spliceosome, and ribosomes. Analysis of the regulatory network involved in the response to drought stress revealed 37 differentially expressed miRNA-mRNA pairs.

Conclusion: This is the first study to simultaneously profile the expression patterns of mRNAs and miRNAs on a genome-wide scale to elucidate the molecular mechanisms of the drought stress responses of S. tonkinensis. Our results suggest that S. tonkinensis implements diverse mechanisms to modulate its responses to drought stress.

Keywords: Drought stress; Next-generation sequencing; Sophora tonkinensis; Transcription factor; mRNA; miRNA.

Fig. 1

S. tonkinensis phenotypic responses to different irrigation treatments. Morphological traits of S. tonkinensis under three irrigation treatments: control treatment (CK), mild drought treatment (MDT), and severe drought treatment (SDT) (A). Bar = 20 mm. The effects of drought treatment on root fresh weight (g) and dry weight (g) in CK, MDT, and SDT (B). Effect of drought treatment on soluble sugar content (C), soluble protein content (D), MDA content (E), peroxidase activity (F), superoxide dismutase activity (G), and catalase activity (H). Each bar represents the mean ± standard error, n = 3. ** indicates a significant difference at p < 0.01 compared with CK using a two-tailed Student’s t-test. Different letters represent significant differences at p < 0.05 (LSD test)

Fig. 2

Unigene annotation results. Annotation information obtained from eight different databases (A). Annotation information based on the Gene Ontology database (B). The most common KEGG pathways involved more than 150 unigenes. The top 19 out of 129 KEGG pathways are presented in this Fig. (C)

Fig. 3

Analysis of differential gene expression and KEGG enrichment. Differentially regulated genes under mild drought treatment (MDT) (A) and severe drought treatment (SDT) (C) compared with the control condition (CK). KEGG enrichment analysis of the DEGs under mild drought treatment (B) and severe drought treatment (D). Venn diagrams showing the numbers of specific and common upregulated (E) and downregulated (G) unigenes. KEGG enrichment analysis of the co-upregulated (F) and co-downregulated (H) unigenes

Fig. 4

Hierarchical clustering of 35 significantly differentially expressed TFs under mild or severe drought treatment. CK: control; MDT: mild drought treatment; SDT: severe drought treatment

Fig. 5

Hierarchical clustering of StLDCs and StCAOs in the control, mild and severe drought treatments. CK: control; MDT: mild drought treatment; SDT: severe drought treatment

Fig. 6

Differentially expressed miRNAs and KEGG annotation of target unigenes. Hierarchical clustering of DEMs in MDT (A) and SDT (B). KEGG annotation of target unigenes of DEMs in MDT (C) and SDT (D)

Fig. 7

Regulatory network between miRNAs and target mRNAs associated with drought stress in MDT (A) and SDT (B). Red circles represent upregulated mRNAs, and blue circles represent downregulated mRNAs. Red diamonds represent upregulated miRNAs, and blue diamonds represent downregulated miRNAs

Fig. 8

Quantitative real-time PCR (qRT-PCR) validation of selected unigenes and miRNAs. A qRT-PCR was performed to determine the expression levels of 11 unigenes relative to actin. B The expression of 6 known miRNAs and 2 novel miRNAs was verified by stem-loop qRT-PCR with 18S RNA as an internal reference gene. Error bars indicate ±SD (n = 3, from three technical replicates). * indicates a significant difference at p < 0.05 compared with CK using a two-tailed Student’s t-test