A Genome-Wide Characterization of New and Drought Stress Responsive MicroRNAs in Populus euphratica
ABSTRACT: We present an efficient method to genome-wide discover new and drought stress responsive miRNAs in P. euphratica. High throughput sequencing of P. euphratica leaves found 197 conserved miRNAs between P. euphratica and Populus trichocarpa. Meanwhile, 189 new miRNAs which belonged to 120 families were identified, a large increasing to the number of P. euphratica miRNAs. Target prediction and degradome sequencing verification of 22 new and 21 conserved miRNA targets showed these targets were involved in multiple biological processes, including transcription regulation and response to stimulus. Furthermore, comparison of high-throughput sequencing with miRNA microarray profiling data indicated that 104 miRNA sequences were up-regulated, while 27 were down-regulated under drought stress. This preliminary characterization based on our findings provided a framework for future analysis of miRNA genes and their roles in key traits of poplar as stress resistance plant breeding and environment protection usage. Overall design: Examination of sRNA expression in 2 poplar leaf samples in drought and normal growth conditions.
Project description:We present an efficient method to genome-wide discover new and drought stress responsive miRNAs in P. euphratica. High throughput sequencing of P. euphratica leaves found 197 conserved miRNAs between P. euphratica and Populus trichocarpa. Meanwhile, 189 new miRNAs which belonged to 120 families were identified, a large increasing to the number of P. euphratica miRNAs. Target prediction and degradome sequencing verification of 22 new and 21 conserved miRNA targets showed these targets were involved in multiple biological processes, including transcription regulation and response to stimulus. Furthermore, comparison of high-throughput sequencing with miRNA microarray profiling data indicated that 104 miRNA sequences were up-regulated, while 27 were down-regulated under drought stress. This preliminary characterization based on our findings provided a framework for future analysis of miRNA genes and their roles in key traits of poplar as stress resistance plant breeding and environment protection usage. Examination of sRNA expression in 2 poplar leaf samples in drought and normal growth conditions.
Project description:BACKGROUND: MicroRNAs (miRNAs) are endogenous small RNAs (sRNAs) with a wide range of regulatory functions in plant development and stress responses. Although miRNAs associated with plant drought stress tolerance have been studied, the use of high-throughput sequencing can provide a much deeper understanding of miRNAs. Drought is a common stress that limits the growth of plants. To obtain more insight into the role of miRNAs in drought stress, Illumina sequencing of Populus trichocarpa sRNAs was implemented. RESULTS: Two sRNA libraries were constructed by sequencing data of control and drought stress treatments of poplar leaves. In total, 207 P. trichocarpa conserved miRNAs were detected from the two sRNA libraries. In addition, 274 potential candidate miRNAs were found; among them, 65 candidates with star sequences were chosen as novel miRNAs. The expression of nine conserved miRNA and three novel miRNAs showed notable changes in response to drought stress. This was also confirmed by quantitative real time polymerase chain reaction experiments. To confirm the targets of miRNAs experimentally, two degradome libraries from the two treatments were constructed. According to degradome sequencing results, 53 and 19 genes were identified as targets of conserved and new miRNAs, respectively. Functional analysis of these miRNA targets indicated that they are involved in important activities such as the regulation of transcription factors, the stress response, and lipid metabolism. CONCLUSIONS: We discovered five upregulated miRNAs and seven downregulated miRNAs in response to drought stress. A total of 72 related target genes were detected by degradome sequencing. These findings reveal important information about the regulation mechanism of miRNAs in P. trichocarpa and promote the understanding of miRNA functions during the drought response.
Project description:BACKGROUND: MicroRNAs (miRNAs) are small, endogenous RNAs that play important regulatory roles in development and stress response in plants by negatively affecting gene expression post-transcriptionally. Identification of miRNAs at the global genome-level by high-throughout sequencing is essential to functionally characterize miRNAs in plants. Drought is one of the common environmental stresses limiting plant growth and development. To understand the role of miRNAs in response of plants to drought stress, drought-responsive miRNAs were identified by high-throughput sequencing in a legume model plant, Medicago truncatula. RESULTS: Two hundreds eighty three and 293 known miRNAs were identified from the control and drought stress libraries, respectively. In addition, 238 potential candidate miRNAs were identified, and among them 14 new miRNAs and 15 new members of known miRNA families whose complementary miRNA*s were also detected. Both high-throughput sequencing and RT-qPCR confirmed that 22 members of 4 miRNA families were up-regulated and 10 members of 6 miRNA families were down-regulated in response to drought stress. Among the 29 new miRNAs/new members of known miRNA families, 8 miRNAs were responsive to drought stress with both 4 miRNAs being up- and down-regulated, respectively. The known and predicted targets of the drought-responsive miRNAs were found to be involved in diverse cellular processes in plants, including development, transcription, protein degradation, detoxification, nutrient status and cross adaptation. CONCLUSIONS: We identified 32 known members of 10 miRNA families and 8 new miRNAs/new members of known miRNA families that were responsive to drought stress by high-throughput sequencing of small RNAs from M. truncatula. These findings are of importance for our understanding of the roles played by miRNAs in response of plants to abiotic stress in general and drought stress in particular.
Project description:MicroRNAs (miRNAs) play important regulatory roles by targeting mRNAs for cleavage or translational repression. Identification of miRNA targets is essential to better understanding the roles of miRNAs. miRNA targets have not been well characterized in mulberry (Morus alba). To anatomize miRNA guided gene regulation under drought stress, transcriptome-wide high throughput degradome sequencing was used in this study to directly detect drought stress responsive miRNA targets in mulberry. A drought library (DL) and a contrast library (CL) were constructed to capture the cleaved mRNAs for sequencing. In CL, 409 target genes of 30 conserved miRNA families and 990 target genes of 199 novel miRNAs were identified. In DL, 373 target genes of 30 conserved miRNA families and 950 target genes of 195 novel miRNAs were identified. Of the conserved miRNA families in DL, mno-miR156, mno-miR172, and mno-miR396 had the highest number of targets with 54, 52 and 41 transcripts, respectively, indicating that these three miRNA families and their target genes might play important functions in response to drought stress in mulberry. Additionally, we found that many of the target genes were transcription factors. By analyzing the miRNA-target molecular network, we found that the DL independent networks consisted of 838 miRNA-mRNA pairs (63.34%). The expression patterns of 11 target genes and 12 correspondent miRNAs were detected using qRT-PCR. Six miRNA targets were further verified by RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-5' RACE). Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these target transcripts were implicated in a broad range of biological processes and various metabolic pathways. This is the first study to comprehensively characterize target genes and their associated miRNAs in response to drought stress by degradome sequencing in mulberry. This study provides a framework for understanding the molecular mechanisms of drought resistance in mulberry.
Project description:MicroRNA (miRNA) is a type of non-coding small RNA with a regulatory function at the posttranscriptional level in plant growth development and in response to abiotic stress. Previous studies have not reported on miRNAs responses to the phytohormone abscisic acid (ABA) at a genome-wide level in Populus euphratica, a model tree for studying abiotic stress responses in woody plants. Here we analyzed the miRNA response to ABA at a genome-wide level in P. euphratica utilizing high-throughput sequencing. To systematically perform a genome-wide analysis of ABA-responsive miRNAs in P. euphratica, nine sRNA libraries derived from three groups (control, treated with ABA for 1 day and treated with ABA for 4 days) were constructed. Each group included three libraries from three individual plantlets as biological replicate. In total, 151 unique mature sequences belonging to 75 conserved miRNA families were identified, and 94 unique sequences were determined to be novel miRNAs, including 56 miRNAs with miRNA(*) sequences. In all, 31 conserved miRNAs and 31 novel miRNAs response to ABA significantly differed among the groups. In addition, 4132 target genes were predicted for the conserved and novel miRNAs. Confirmed by real-time qPCR, expression changes of miRNAs were inversely correlated with the expression profiles of their putative targets. The Populus special or novel miRNA-target interactions were predicted might be involved in some biological process related stress tolerance. Our analysis provides a comprehensive view of how P. euphratica miRNA respond to ABA, and moreover, different temporal dynamics were observed in different ABA-treated libraries.
Project description:Drought stress is a major obstacle to agricultural production. Tibetan wild barley with rich genetic diversity is useful for drought-tolerant improvement of cereals. MicroRNAs (miRNAs) play critical roles in controlling gene expression in response to various environment perturbations in plants. However, the genome-wide expression profiles of miRNAs and their targets in response to drought stress are largely unknown in wild barley. In this study, a polyethylene glycol (PEG) induced drought stress hydroponic experiment was performed, and the expression profiles of miRNAs from the roots of two contrasting Tibetan wild barley genotypes XZ5 (drought-tolerant) and XZ54 (drought-sensitive), and one cultivated barley Tadmor (drought-tolerant) generated by high-throughput sequencing were compared. There were 69 conserved miRNAs and 1574 novel miRNAs in the dataset of three genotypes under control and drought conditions. Among them, seven conserved miRNAs and 36 novel miRNAs showed significantly genotype-specific expression patterns in response to drought stress. And 12 miRNAs were further regarded as drought tolerant associated miRNAs in XZ5, which mostly participate in gene expression, metabolism, signaling and transportation, suggesting that they and their target genes play important roles in plant drought tolerance. This is the first comparation study on the miRNA transcriptome in the roots of two Tibetan wild barley genotypes differing in drought tolerance and one drought tolerant cultivar in response to PEG treatment. Further results revealed the candidate drought tolerant miRNAs and target genes in the miRNA regulation mechanism in wild barley under drought stress. Our findings provide valuable understandings for the functional characterization of miRNAs in drought tolerance.
Project description:Alfalfa (Medicago sativa L.) is a high quality leguminous forage. Drought stress is one of the main factors that restrict the development of the alfalfa industry. High-throughput sequencing was used to analyze the microRNA (miRNA) profiles of alfalfa plants treated with CK (normal water), PEG (polyethylene glycol-6000; drought stress), and PEG + SNP (sodium nitroprusside; nitric oxide (NO) sprayed externally under drought stress). We identified 90 known miRNAs belonging to 46 families and predicted 177 new miRNAs. Real-time quantitative fluorescent PCR (qRT-PCR) was used to validate high-throughput expression analysis data. A total of 32 (14 known miRNAs and 18 new miRNAs) and 55 (24 known miRNAs and 31 new miRNAs) differentially expressed miRNAs were identified in PEG and PEG + SNP samples. This suggested that exogenous NO can induce more new miRNAs. The differentially expressed miRNA maturation sequences in the two treatment groups were targeted by 86 and 157 potential target genes, separately. The function of target genes was annotated by gene ontology (GO) enrichment and kyoto encyclopedia of genes and genomes (KEGG) analysis. The expression profiles of nine selected miRNAs and their target genes verified that their expression patterns were opposite. This study has documented that analysis of miRNA under PEG and PEG + SNP conditions provides important insights into the improvement of drought resistance of alfalfa by exogenous NO at the molecular level. This has important scientific value and practical significance for the improvement of plant drought resistance by exogenous NO.
Project description:BACKGROUND: Populus euphratica is a representative model woody plant species for studying resistance to abiotic stresses such as drought and salt. Salt stress is one of the most common environmental factors that affect plant growth and development. MicroRNAs (miRNAs) are small, noncoding RNAs that have important regulatory functions in plant growth, development, and response to abiotic stress. RESULTS: To investigate the miRNAs involved in the salt-stress response, we constructed four small cDNA libraries from P. euphratica plantlets treated with or without salt (300 mM NaCl, 3 days) in either the root or leaf. Using high-throughput sequencing to identify miRNAs, we found 164 conserved miRNAs belonging to 44 families. Of these, 136 novel miRNAs were from the leaf, and 128 novel miRNAs were from the root. In response to salt stress, 95 miRNAs belonging to 46 conserved miRNAs families changed significantly, with 56 miRNAs upregulated and 39 miRNAs downregulated in the leaf. A comparison of the leaf and root tissues revealed 155 miRNAs belonging to 63 families with significantly altered expression, including 84 upregulated and 71 downregulated miRNAs. Furthermore, 479 target genes in the root and 541 targets of novel miRNAs in the leaf were predicted, and functional information was annotated using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. CONCLUSIONS: This study provides a novel visual field for understanding the regulatory roles of miRNAs in response to salt stress in Populus.
Project description:BACKGROUND: In plants, microRNAs (miRNAs) are endogenous ~22 nt RNAs that play important regulatory roles in many aspects of plant biology, including metabolism, hormone response, epigenetic control of transposable elements, and stress response. Extensive studies of miRNAs have been performed in model plants such as rice and Arabidopsis thaliana. In maize, most miRNAs and their target genes were analyzed and identified by clearly different treatments, such as response to low nitrate, salt and drought stress. However, little is known about miRNAs involved in maize ear development. The objective of this study is to identify conserved and novel miRNAs and their target genes by combined small RNA and degradome sequencing at four inflorescence developmental stages. RESULTS: We used deep-sequencing, miRNA microarray assays and computational methods to identify, profile, and describe conserved and non-conserved miRNAs at four ear developmental stages, which resulted in identification of 22 conserved and 21-maize-specific miRNA families together with their corresponding miRNA*. Comparison of miRNA expression in these developmental stages revealed 18 differentially expressed miRNA families. Finally, a total of 141 genes (251 transcripts) targeted by 102 small RNAs including 98 miRNAs and 4 ta-siRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs. Moreover, the differentially expressed miRNAs-mediated pathways that regulate the development of ears were discussed. CONCLUSIONS: This study confirmed 22 conserved miRNA families and discovered 26 novel miRNAs in maize. Moreover, we identified 141 target genes of known and new miRNAs and ta-siRNAs. Of these, 72 genes (117 transcripts) targeted by 62 differentially expressed miRNAs may attribute to the development of maize ears. Identification and characterization of these important classes of regulatory genes in maize may improve our understanding of molecular mechanisms controlling ear development.
Project description:The study of microRNAs (miRNAs) in plants has gained significant attention in recent years due to their regulatory role during development and in response to biotic and abiotic stresses. Although cassava (Manihot esculenta Crantz) is tolerant to drought and other adverse conditions, most cassava miRNAs have been predicted using bioinformatics alone or through sequencing of plants challenged by biotic stress. Here, we use high-throughput sequencing and different bioinformatics methods to identify potential cassava miRNAs expressed in different tissues subject to heat and drought conditions. We identified 60?miRNAs conserved in other plant species and 821 potential cassava-specific miRNAs. We also predicted 134 and 1002 potential target genes for these two sets of sequences. Using real time PCR, we verified the condition-specific expression of 5 cassava small RNAs relative to a non-stress control. We also found, using publicly available expression data, a significantly lower expression of the predicted target genes of conserved and nonconserved miRNAs under drought stress compared to other cassava genes. Gene Ontology enrichment analysis along with condition specific expression of predicted miRNA targets, allowed us to identify several interesting miRNAs which may play a role in stress-induced posttranscriptional regulation in cassava and other plants.