High-Throughput Sequencing Reveals H2O2 Stress-Associated MicroRNAs and a Potential Regulatory Network in Brachypodium distachyon Seedlings

Abstract

Oxidative stress in plants can be triggered by many environmental stress factors, such as drought and salinity. Brachypodium distachyon is a model organism for the study of biofuel plants and crops, such as wheat. Although recent studies have found many oxidative stress response-related proteins, the mechanism of microRNA (miRNA)-mediated oxidative stress response is still unclear. Using next generation high-throughput sequencing technology, the small RNAs were sequenced from the model plant B. distachyon 21 (Bd21) under H₂O₂ stress and normal growth conditions. In total, 144 known B. distachyon miRNAs and 221 potential new miRNAs were identified. Further analysis of potential new miRNAs suggested that 36 could be clustered into known miRNA families, while the remaining 185 were identified as B. distachyon-specific new miRNAs. Differential analysis of miRNAs from the normal and H₂O₂ stress libraries identified 31 known and 30 new H₂O₂ stress responsive miRNAs. The expression patterns of seven representative miRNAs were verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, which produced results consistent with those of the deep sequencing method. Moreover, we also performed RT-qPCR analysis to verify the expression levels of 13 target genes and the cleavage site of 5 target genes by known or novel miRNAs were validated experimentally by 5' RACE. Additionally, a miRNA-mediated gene regulatory network for H₂O₂ stress response was constructed. Our study identifies a set of H₂O₂-responsive miRNAs and their target genes and reveals the mechanism of oxidative stress response and defense at the post-transcriptional regulatory level.

Keywords: Bd21; H2O2 stress; high-throughput sequencing; microRNA; regulatory network.

Figure 1

Length distribution of sRNAs detected in the control library and stress library. (A) Redundant sRNAs; (B) unique sRNAs.

Figure 2

Known and novel miRNAs differentially expressed between the CS library and TS library. (A) Known miRNAs; (B) novel miRNAs.

Figure 3

GO enrichment of the H₂O₂-responsive miRNA target genes. The dataset containing protein sequences of B. distachyon genome was set as the background dataset. The vertical axis is the enriched GO category, and the horizontal axis is GO enrichment.

Figure 4

A miRNA-mediated H₂O₂-responsive regulatory network. Triangles represent the differentially expressed miRNAs and the color gradient shows the fold change (red: upregulation; green: downregulation). Blue circles represent the target genes of differentially expressed miRNAs; yellow circles represent target genes that encode transcription factors.

Figure 5

RT-qPCR validation of the miRNAs. Blue bar: relative gene expression level in the control library. Red bar: relative gene expression level in the H₂O₂ stress library. The data are derived from three biological repeats and represent mean ± standard deviation (n = 3). “^*” and “^**” indicate significant difference at P < 0.05 and 0.01 level, respectively.

Figure 6

RT-qPCR analysis of the miRNA target genes. The Actin and SamDC genes were served as the endogenous controls. Blue bar: relative gene expression level in the control library. Red bar: relative gene expression level in the H₂O₂ stress library. The data are derived from three biological repeats and represent mean ± standard deviation (n = 3). “^*” and “^**” indicate significant difference at P < 0.05 and 0.01 level, respectively.

Figure 7

Validation of known and novel miRNAs by RLM-5′-RACE. Each top strand represents the miRNA complementary site on target mRNA, and each bottom strand represents the miRNA. Watson-Crick pairing (vertical dashes) and G:U wobble pairing (circles) are indicated. The arrows indicated the cleavage sites of target genes, and the numbers showed the frequency of cloned 5′ RACE products.