Transcriptomic analysis reveals the regulatory mechanisms of messenger RNA (mRNA) and long non-coding RNA (lncRNA) in response to waterlogging stress in rye (Secale cereale L.)

Abstract

Background: Waterlogging stress (WS) negatively impacts crop growth and productivity, making it important to understand crop resistance processes and discover useful WS resistance genes. In this study, rye cultivars and wild rye species were subjected to 12-day WS treatment, and the cultivar Secale cereale L. Imperil showed higher tolerance. Whole transcriptome sequencing was performed on this cultivar to identify differentially expressed (DE) messenger RNAs (DE-mRNAs) and long non-coding RNAs (DE-lncRNAs) involved in WS response.

Results: Among the 6 species, Secale cereale L. Imperil showed higher tolerance than wild rye species against WS. The cultivar effectively mitigated oxidative stress, and regulated hydrogen peroxide and superoxide anion. A total of 728 DE-mRNAs and 60 DE-lncRNAs were discovered. Among these, 318 DE-mRNAs and 32 DE-lncRNAs were upregulated, and 410 DE-mRNAs and 28 DE-lncRNAs were downregulated. GO enrichment analysis discovered metabolic processes, cellular processes, and single-organism processes as enriched biological processes (BP). For cellular components (CC), the enriched terms were membrane, membrane part, cell, and cell part. Enriched molecular functions (MF) terms were catalytic activity, binding, and transporter activity. LncRNA and mRNA regulatory processes were mainly related to MAPK signaling pathway-plant, plant hormone signal transduction, phenylpropanoid biosynthesis, anthocyanin biosynthesis, glutathione metabolism, ubiquitin-mediated proteolysis, ABC transporter, Cytochrome b6/f complex, secondary metabolite biosynthesis, and carotenoid biosynthesis pathways. The signalling of ethylene-related pathways was not mainly dependent on AP2/ERF and WRKY transcription factors (TF), but on other factors. Photosynthetic activity was active, and carotenoid levels increased in rye under WS. Sphingolipids, the cytochrome b6/f complex, and glutamate are involved in rye WS response. Sucrose transportation was not significantly inhibited, and sucrose breakdown occurs in rye under WS.

Conclusions: This study investigated the expression levels and regulatory functions of mRNAs and lncRNAs in 12-day waterlogged rye seedlings. The findings shed light on the genes that play a significant role in rye ability to withstand WS. The findings from this study will serve as a foundation for further investigations into the mRNA and lncRNA WS responses in rye.

Keywords: DE-lncRNA; DE-mRNA; Rye (Secale cereale L.); Transcriptome sequencing; Waterlogging stress (WS).

Fig. 1

Treatment and physiological parameters. Waterlogging stress (WT) and control (CK) seedlings of four rye cultivars and two wild species (A), malondialdehyde content (B), and enzymatic antioxidant activities of catalase (C), peroxidase (D), and superoxide dismutase (E) of WT and CK seedlings. Significant differences between three biological replicates are denoted by asterisks, indicating statistical significance at p < 0.05. The levels of significance are indicated as p < 0.001 (**) and p < 0.0005 (***)

Fig. 2

Gene expression and enrichment annotation of DE-mRNAs. Expressed DE-mRNAs (A), GO classification annotation of DE-mRNAs (B), KEGG pathway classification enrichment of DE-mRNAs (C), and annotated gene expression regulatory proteins and transcriptional factors of up-regulated DE-mRNAs (D)

Fig. 3

Expression, characterization, and network analysis of lncRNAs. Classification of lncRNAs (A), hierarchical clustering of DE-lncRNAs (B), classification of lncRNAs in the reference genome: sense-lncRNAs are shown by the green colour; red represents intergenic-lncRNAs; blue represents intronic-lncRNAs; and black represents antisense-lncRNAs (C), and the protein-protein interaction network of DE-lncRNA target genes (D)

Fig. 4

Comparative analysis of lncRNAs and mRNAs. Transcript length distribution (A), number of exons (B), length of ORF (C), alternative splicing event isoform diversity (D), and visualisation of DE-mRNAs and lncRNAs in the reference genome (E): The outer circle represents the chromosomes based on the reference genome; the centre circle depicts the distribution of mRNAs on the chromosomes; and the inner circle represents the distribution of lncRNAs; upregulated DE-mRNAs are shown in red, while downregulated DE-mRNAs are shown in green. Upregulated DE-lncRNAs are highlighted in yellow, and downregulated DE-lncRNAs are highlighted in blue

Fig. 5

Classification and enrichment analysis of DE-lncRNA target genes. GO enrichment annotation of DE-lncRNA target genes (A), KEGG pathway classification annotation of DE-lncRNA target genes (B), and statistics of KEGG pathway enrichment of DE-lncRNA target genes (C)

Fig. 6

Co-expression association of mRNA-expressed genes. Expression module of mRNAs (A) and expression module clusters of mRNAs (B)

Fig. 7

Sequencing validation and motif discovery. RT–qPCR analysis of selected upregulated DE-mRNAs (A), conserved motifs in the exons of the selected DE-mRNAs (B), and cis-acting regulatory elements in the promoter region of the selected DE-mRNAs (C). Asterisks represent significant differences between three biological replicates at p = 0.0005 (***) and p < 0.0001 (****)