Combined analysis of mRNA and miRNA identifies dehydration and salinity responsive key molecular players in citrus roots

Abstract

Citrus is one of the most economically important fruit crops around world. Drought and salinity stresses adversely affected its productivity and fruit quality. However, the genetic regulatory networks and signaling pathways involved in drought and salinity remain to be elucidated. With RNA-seq and sRNA-seq, an integrative analysis of miRNA and mRNA expression profiling and their regulatory networks were conducted using citrus roots subjected to dehydration and salt treatment. Differentially expressed (DE) mRNA and miRNA profiles were obtained according to fold change analysis and the relationships between miRNAs and target mRNAs were found to be coherent and incoherent in the regulatory networks. GO enrichment analysis revealed that some crucial biological processes related to signal transduction (e.g. 'MAPK cascade'), hormone-mediated signaling pathways (e.g. abscisic acid- activated signaling pathway'), reactive oxygen species (ROS) metabolic process (e.g. 'hydrogen peroxide catabolic process') and transcription factors (e.g., 'MYB, ZFP and bZIP') were involved in dehydration and/or salt treatment. The molecular players in response to dehydration and salt treatment were partially overlapping. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-seq and sRNA-seq analysis. This study provides new insights into the molecular mechanisms how citrus roots respond to dehydration and salt treatment.

Figure 1. The correlation between each two samples based on FPKM result.

Figure 2. Length (nt) distribution of sRNAs.

Figure 3. Results from qRT-PCR of miRNAs and mRNAs in Citrus junos.

sRNAs and mRNAs were isolated from roots treated with dehydration and salt, respectively. The expression levels of miRNAs and mRNAs were normalized to U6 snRNA and Actin gene, respectively. The mormalized miRNA and mRNA levels in the control were arbitrarily set to 1.

Figure 4. Functional categorization of significantly differentially expressed genes in Citrus junos roots under dehydration (blue column) and salt stress (red column).

Functional categorization was performed with BGI WEGO.

Figure 5. Pathway enrichment analysis of significantly differentially expressed genes in Citrus junos roots under dehydration and salt stress.

Figure 6. miRNA-mRNA correlation network.

DR and SA indicate dehydration and salt treatment, respectively. Down-regulated mRNAs and miRNAs were shown as green and the up-regulated shown as red.

Figure 7. Mapping of the mRNA cleavage sites by RNA ligase-mediated 5′ RANC.

Watson-Crick pairing was indicated by vertical dashes and G:U wobble paring by circles. The arrows indicated the 5′ termini of mRNA fragments isolated from roots of Citrus junos Siebold cv. ‘Ziyang’, as identified by cloned 5′RACE products, with the frequency of clones shown. RNA ligase-mediated 5′RACE was used to map the cleavage sites. The partial mRNA sequences from the target genes were aligned with the miRNAs.