Integrated analysis of transcriptome and miRNAome reveals the heat stress response of Pinellia ternata seedlings

Abstract

Pinellia ternata (Thunb.) Briet., a valuable herb native to China, is susceptible to the "sprout tumble" phenomenon because of high temperatures, resulting in a significant yield reduction. However, the molecular regulatory mechanisms underlying the response of P. ternata to heat stress are not well understood. In this study, we integrated transcriptome and miRNAome sequencing to identify heat-response genes, microRNAs (miRNAs), and key miRNA-target pairs in P. ternata that differed between heat-stress and room-temperature conditions. Transcriptome analysis revealed extensive reprogramming of 4,960 genes across various categories, predominantly associated with cellular and metabolic processes, responses to stimuli, biological regulation, cell parts, organelles, membranes, and catalytic and binding activities. miRNAome sequencing identified 1,597 known/conserved miRNAs that were differentially expressed between the two test conditions. According to the analysis, genes and miRNAs associated with the regulation of transcription, DNA template, transcription factor activity, and sequence-specific DNA binding pathways may play a major role in the resistance to heat stress in P. ternata. Integrated analysis of the transcriptome and miRNAome expression data revealed 41 high-confidence miRNA-mRNA pairs, forming 25 modules. MYB-like proteins and calcium-responsive transcription coactivators may play an integral role in heat-stress resistance in P. ternata. Additionally, the candidate genes and miRNAs were subjected to quantitative real-time polymerase chain reaction to validate their expression patterns. These results offer a foundation for future studies exploring the mechanisms and critical genes involved in heat-stress resistance in P. ternata.

Keywords: Pinellia ternata; Heat stress; Regulation; Transcriptome; miRNAs.

Fig. 1

Physiological responses of Pinellia ternata seedlings before and after heat treatment. (A) P. ternata seedlings phenotypes in response to heat stress. Plants were grown in soil to the three-leaf stage and subjected to a 40 ℃ stress treatment for 24 h (left, scale bar = 4 cm). DAB and NBT staining from the same part of P. ternata seedlings before and after heat treatment (right, scale bar = 15 mm). (B) MDA and chlorophyll contents, and POD activities in P. ternata seedlings under control and heat treatment for 24 h. Except where noted, all data are presented as mean (n = 3) and standard deviation. Data were analyzed by Student’s t-test and one-way _ANOVA. * and ** indicate significant differences between control and heat treatment plants at the P < 0.05 and P < 0.01 levels, respectively

Fig. 2

Transcriptome sequencing analysis of G and C. (A) scatter plot of DEGs in G vs. C. Significantly differentially expressed genes are represented by red dots (up-regulated) and green dots (down-regulated), whereas non-differentially expressed genes are represented by black dots. (B) Gene ontology (GO) classification of the DEGs in P. ternata seedlings under heat stress. The ordinate is the enriched GO term, and the abscissa is the percent and number of differentially expressed genes in this term. Different colors are used to distinguish biological processes, cellular components, and molecular functions. (C) Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment of the DEGs in P. ternata under heat stress. The ordinate is the enriched KEGG term, and the abscissa is the percent and number of differentially expressed genes in this term. Different colors are used to distinguish biological processes, cellular components, and molecular functions

Fig. 3

Heatmap of the regulatory multiples of 38 transcription factors with different regulatory trends in the comparisons of control and heat stress treatments. Transcription factors of the same family are indicated by squares of the same color, and the number of genes is attached to the gene family name. The colored bars represent the 38 transcription factor values of control and heat treatment [log₂(TPM + 1)]

Fig. 4

Analysis of differentially expressed miRNAs (DEMs) between G and C. (A) The number of DEMs in response to heat stress treatments in P. ternata seedlings. (B) Gene ontology (GO) enrichment analysis of stress-responsive DEMs under heat treatments. The ordinate is the enriched GO term, and the abscissa is the percent and number of differentially expressed genes in this term. Different colors are used to distinguish biological processes, cellular components, and molecular functions

Fig. 5

Validation of expression profile of miRNAs and their predicted target genes in P. ternata seedlings. Expression analysis of selected miRNAs (A) and one of their predicted targets genes (B) using qRT-PCR. Three biological replicates and two technical replicates were included in the study. Asterisks indicate significant differences between the control and the heat treatment sample (Student’s t-test, *P < 0.05, **P < 0.01)

Fig. 6

Molecular regulatory network diagram. The black lines represent the interaction between hub miRNA with their corresponding targeted genes