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

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 havefound 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 wereidentified. Further analysis of potential new miRNAs suggested that 36 could beclustered 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, whichproduced results consistent with those of the deep sequencing method. Moreover, wealso 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 validatedexperimentally 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.