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

Dong-Wen Lv,Yue-Ming Yan,Yan-Wei Bian,Cai-Xia Han,Guan-Xing Chen,Zi-Tong Yu,Geng-Rui Zhu,Shoumin Zhen

doi:10.3389/fpls.2016.01567

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.

Highlights

Many environmental stress factors, including high light, UV irradiation, heat, salinity, drought, and cold, can cause plant cells to produce reactive oxygen species (ROS), leading to acceleration of lipid peroxidation and leaf senescence (Mittler et al, 2004; Upadhyaya et al, 2007)
In contrast to other ROS, such as O−2 and hydroxyl radicals (OH−), Hydrogen peroxide (H2O2) can pass through membranes (Foyer et al, 1997; Uchida et al, 2002; de Azevedo Neto et al, 2005; Wahid et al, 2007) and is relatively stable, so it is suitable for its roles as an important component of cell signaling cascades
In this study, we identified miRNAs and their potential target genes related to H2O2 stress using high-throughput sequencing, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and 5′ RACE, combined with bioinformatics methods

Summary

Introduction

Many environmental stress factors, including high light, UV irradiation, heat, salinity, drought, and cold, can cause plant cells to produce reactive oxygen species (ROS), leading to acceleration of lipid peroxidation and leaf senescence (Mittler et al, 2004; Upadhyaya et al, 2007). In contrast to other ROS, such as O−2 and hydroxyl radicals (OH−), H2O2 can pass through membranes (Foyer et al, 1997; Uchida et al, 2002; de Azevedo Neto et al, 2005; Wahid et al, 2007) and is relatively stable, so it is suitable for its roles as an important component of cell signaling cascades At low concentrations H2O2 may play a role as a signaling molecule, whereas at high concentrations H2O2 will cause programmed cell death (Quan et al, 2008)

Methods

Findings

Discussion

Conclusion