Identification of Radish (Raphanus sativus L.) miRNAs and Their Target Genes to Explore miRNA-Mediated Regulatory Networks in Lead (Pb) Stress Responses by High-Throughput Sequencing and Degradome Analysis

Abstract

Increasing evidence has revealed that microRNA (miRNA)-mediated gene regulation plays a significant role in response to heavy metal stresses. However, there is little information available about the expression patterns or roles of miRNAs under lead toxicity stress in plants. The radish is an important root vegetable crop with a fleshy taproot as the edible part. It was of vital importance to investigate the response mechanisms and explore the regulatory network at the molecular level under the heavy metal stresses in radish. In the present study, using high-throughput sequencing and degradome analysis, a genome-wide identification of radish miRNA and their targets under the exposure of Pb stress was conducted. A total of 74 known and 173 potential novel miRNAs were successfully identified from two radish root libraries of one untreated control (CK) and one Pb-stressed (Pb500). Of these, 25 known and nine novel miRNAs were significantly differentially expressed and identified as Pbresponsive miRNAs. Degradome analysis revealed that 1,979 miRNA-mRNA target transcripts could potentially be cleaved. Gene Ontology (GO) analysis revealed that these target transcripts were predominately involved in the regulation of transcription, defense responses, and binding related terms. The identified target genes for Pb-responsive miRNAs were mainly involved in stress-related signal sensing and transduction, specific metal uptake and homeostasis mechanisms. Additionally, the expression patterns of 20 Pbresponsive miRNAs and six target genes were validated by quantitative real-time PCR (qRT-PCR). These results provide fundamental insights into the miRNA-mediated regulatory networks and molecular mechanisms underlying plant responsiveness to Pb stresses.

Transcriptome-wide analysis of chromium-stress responsive microRNAs to explore miRNA-mediated regulatory networks in radish (Raphanus sativus L.)

“…Plant responses to metal toxicity exhibit various physiological and biochemical processes that require fine and precise regulation at transcriptional and posttranscriptional levels 25 . Recently, a number of miRNAs and their corresponding targets have been comprehensively identified using high-throughput Solexa sequencing technology in some important plant species 7 26 27 , which proved to be involved in plant response to HM stresses including Cd 13 28 , Al 21 22 , As 19 , Hg 14 and Pb 29 . However, few studies on extensive identification of Cr-responsive miRNAs and their target genes have yet been reported in vegetable crops.…”

“…The characterization of potential targets for stress related miRNAs is a critical step for clarification of miRNA-mediated regulatory networks associated with plant response to HM stresses 29 34 . Recently, some studies have been conducted on the identification of various targets for HM-responsive miRNAs, which made great contributions to the understanding of the critical involvement of miRNAs and their target genes in HM toxicity responses in plants 20 34 .…”

“…In this study, a number of key HM-responsive enzymes or proteins mediating the HM uptake, accumulation, translocation and detoxification processes in plants, were identified as target transcripts for several known and novel miRNAs. The yellow stripe-like 1 gene ( YSL1 ) was cleaved by miR5265 and could play a pivotal role in HM homeostasis in plants, which functioned as a metal-nicotianamine (NA) transporter for metal ion transport 29 44 . In addition, five genes encoding two ABC transporter proteins (PDR5 and PXA1), one heavy metal ATPase (HMA5) and two HM transport/detoxification domain-containing proteins, which were considered as indispensible ingredients in metal uptake and translocation in plants 13 28 , were identified for miR7767-3p, rsa-miRn55, miR5671, rsa-miRn44 and miR4993, respectively.…”

Identification of bolting-related microRNAs and their targets reveals complex miRNA-mediated flowering-time regulatory networks in radish (Raphanus sativus L.)

“…Many plant miRNAs are evolutionally conserved among many species 8 9 . Recent evidences have indicated that miRNAs could play pivotal roles in various plant growth and developmental processes, such as hormone homeostasis 10 11 , root development 12 13 , leaf morphogenesis 14 15 , flower development 11 16 17 , embryogenesis 18 19 and stress responses 20 21 .…”

Toxic-Metal-Induced Alteration in miRNA Expression Profile as a Proposed Mechanism for Disease Development

“…Another experiment performed on Raphanus sativus identified 25 known and 9 novel Pb-responsive miRNAs primarily involved in the stress-related signal, metal absorption, and homeostasis mechanisms. The authors observed the downregulation of PN-miRNAc1, which plays a role in ROS signaling and synthesis of the glutathione S-transferase [105]. Additionally, numerous miRNAs were identified in plants after exposure to Pb.…”