- drought tolerance and productivity in rice. - The drought-responsive miRNAs (DRMs) formed complex regulatory network via their coexpression and direct/indirect impacts on the rice transcriptome. - miRNAs have profound impacts on the rice transcriptome reflected by great number of correlated drought-responsive genes. - Conclusion: Based on the temporal pattern of miRNAs in response to drought, we have described the complex network between DRMs. - This knowledge provides valuable information for a better understanding in the roles of miRNAs play in rice DT and/or GDP, which can facilitate our utilization of miRNA in breeding.. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - This requires improved understanding of the association of a miRNA with stress-tolerance and/or GDP.. - Recently, attentions have been focused on the importance of posttranscriptional regulation by miRNAs in drought tolerance (DT) due to their central roles in the regulatory network [7, 38]. - With the fast develop- ment of next-generation sequencing, drought-responsive miRNAs (DRMs) have been identified in diverse crops, including cotton [48], rapeseed [21], maize [1], tomato [28], and rice (Oryza sativa) [3, 5, 6]. - Many DRMs have been characterized as important modulators in DT via regulating the expression of drought-responsive genes [7]. - The improvement of DT in rice is thus a primary breeding aim for “green super rice” [31, 52]. - For this purpose, the roles played by miRNAs in rice drought-resistance have been widely studied. - For better utilization of miRNAs, it is necessary to understand their associations with DT and/or GDP in rice.. - To understand the potential roles played by miRNAs in rice DT, we investigated the genome-wide expression of miRNAs in six rice genotypes at five time points under drought stress and one time point at recovery. - Table S2) and dead leaves (Additional file: Table S1) in the rice genotypes. - To resist drought, the rice acti- vated mechanisms of osmotic adjustment and ROS scavenging, as reflected in the largely increased os- motic potential (Additional file: Table S3) and total antioxidant capacity (Additional file 1: Table S4) under drought conditions, particularly in later drought time points (D3-D5).. - General description of drought-responsive and recovery- related miRNAs detected in the six rice genotypes A total of 632 conserved mature miRNAs in miRBase were detected in 66 sequenced samples (Additional file 1:. - During the drought period, 354 miRNAs in 57 families were identified as drought-responsive miRNAs (DRMs). - How- ever, 107 DRMs could be frequently (frequency ≥ 3) de- tected among different genotypes and time points (Additional file 2: Figure S5a), suggesting that they have universal roles in rice adaptation to drought. - indicated complicated regulatory networks of miRNAs in response to drought.. - Correlations of miRNAs with GDP and DT traits. - 2 A heatmap of the matrix of Pearson ’ s correlation coefficient among drought-responsive miRNAs based on their expressions. - The prediction based on the correlation analysis was partially validated by the miRNAs that have been functionally characterized in rice (Additional file 1: Table S . - Interestingly, DRMs of the same type were more generally highly cor- related (Fig. - 3), indicating its positive role in rice DT.. - Time course of the regulation of miRNAs. - This indicated that DRMs in cluster-2 might be associated with DT in the late stage. - This indicated that DRMs in cluster-2 might be associated with DT in the early stage. - 4 A heatmap of time-series regulations of drought-responsive miRNAs (DRMs) during drought period. - In particular, 143 DRMs and 7 for types I, II, III, and IV, respectively) in cluster-5b exhibited greater fold changes than DRMs in the cluster-5a and were gradually upregulated throughout the period of drought, indicating that they have important roles in rice DT.. - As expected, the expression of LOC_Os12g40890 was increased in the overexpression lines of pre- OsmiR408 (Additional file 2: Figure S6), which poten- tially regulated DT in the transgenic lines. - Additional file 3). - DRMs of type II tended to be involved in the generation of precursor metabolites and energy, photosynthesis, lipid metabolic process, transport, cell death, biosynthetic process, anatomical structure morpho- genesis, and post-embryonic development. - Time course of the regulation of miRNAs under drought conditions provides key information to identify miRNAs associated with DT. - miRNAs are in the response to drought stress and play essential roles in rice DT [7, 49]. - The common design of these experiments is to generate drought-responsive miRNAs from a single genotype or a pair of genotypes with contrasting toler- ance [3, 5]. - The involvement of a limited number of genotypes and time points when investigating DRGs may result in the loss of valuable information [17]. - By time course investigation of miRNAs in response to drought among six rice genotypes, we identified greater. - number of conserved drought-responsive miRNAs than previous studies (354 mature miRNA in 57 families).. - Their activations limited in the early drought may bring less penalties on GDP.. - Complex regulatory networks of miRNAs in rice DT and/. - First, we detected a complex regulatory network of DRMs based on the correlation analysis. - We found that DRMs of the same type worked together to activate the tolerant mechanisms and/or to inhibit rice GDP. - Given the central role of miRNAs in the regulatory network [7, 38], a DT-associated miRNA can have pleiotropic effects on GDP [38, 46]. - As reported by two independent studies, overexpression of pre-OsmiRNA408 in rice decreases rice drought tolerance [37] while it posi- tively regulates grain yield [50]. - These miRNAs have great prospects in developing DT in rice.. - In this study, we detected 344 drought responsive miR- NAs among six rice genotypes in total and described their temporal regulations along a progressive drought.. - The depth of the soil layer in the drought-treated field was limited to 30 cm. - Moreover, planting rice in the experimental field rather than in pots led to more homogenous levels [32]. - Three replicates were set up for each geno- type in the drought-treated and well-watered fields. - We measured the soil water content at a depth of 30 cm in the drought-treated field every 3–5 days to monitor the progress of the drought. - The drought-treated field was re-watered on the afternoon of the 22nd of August until the SWC decreased to ~ 10%. - Osmolality were measured to reflect the osmotic adjustment of the leaf samples. - The expression data of the available miRNAs from each sample are provided in. - Additional file 4. - The drought-responsive miRNA (DRMs) and recovery-related miRNAs (RRMs) for each genotype were determined using p-value<. - We then as- sembled the clean data using the software Cufflinks and mapped them to the reference genome (Nipponbare, msu7.0) and to mitochondrial and chloroplast genomes (http://rice.plantbiology.msu.edu/) via Tophat with no more than two base mismatches allowed in the align- ment [41]. - Studying impacts of miRNAs on the rice transcriptome, GDP, and DT by correlation analyses. - To construct the regulatory network of the DRMs, cor- relation analyses (Pearson’s correlation coefficient, PCC) were applied to detect any significant correlations among DRMs based on their expressions. - De- tailed information can be found in the China Rice Data Center (http://www.ricedata.cn/gene. - DRMs and DEGs (from D1-D5 and R) to study the in- direct impacts of miRNAs on the rice transcriptome.. - Based on the results of the correlation analysis, four types of DRMs were defined: (1) Type I, a DRM highly correlated with at least one GDP trait. - 30 highly corre- lated DRGs, it was not included in the GO and KEGG enrichment analyses. - Fold changes in the expression of each miRNA in the six genotypes from a single time point were averaged in this analysis. - Based on this method, miRNAs could be divided into five major clusters based on the time course regulation of the miRNAs. - Number of expressed, available, drought- responsive miRNAs (DRMs), and recovery-related miRNAs (RRMs). - Validation of expression patterns and correlation-based predictions by former functionally characterized miRNAs in rice. - Target genes predicted by TargetFinder and psRobot for drought-responsive miRNAs. - Number of predicted target genes of differentially expressed miRNAs in drought responsive genes (DRGs) with different Pearson ’ s Correlation Coefficient (PCC) values. - List of GDP (growth, development, and reproduction)- and DT (drought-tolerance)-associated drought-responsive. - Distribution of the clean reads in small RNA libraries. - A heatmap of the frequency of genotypes for drought- responsive miRNAs during drought (D1-D5). - A heatmap of the frequency of time points (D1-D5) for drought-responsive miRNAs among six genotypes. - Summary of drought-responsive miR- NAs (DRMs) and recovery-related miRNAs (RRMs). - A heatmap describing the involvements of DRMs in biological processes based on the Gene Ontology enrichment using their highly correlated drought-responsive genes (|PCC >. - A heat- map describing the involvements of DRMs in metabolic pathways based on the KEGG enrichment using their highly correlated drought- responsive genes (|PCC >. - -0.4) correlated drought-responsive genes (DRGs) for OsmiR1870-3p and OsmiR1870-5p. - 0.05 in the enrichment analyses, respectively. - Fold changes (drought/ well-watered) of six drought- responsive miRNAs quantified by high-throughput sequencing is well val- idated by qPCR. - Correlations between expressions of six drought responsive genes quantified by RNA-seq and qPCR. - The data can be also found in the reference Ma et al. - Additional file 3. - Matrix of Pearson ’ s correlation coefficients (PCCs) between drought-responsive miRNAs and drought-responsive genes.. - Additional file 5. - Expressions (by FPKM) of drought-responsive genes.. - DRM: Drought-responsive miRNA. - Identification, characterization, and functional validation of drought-responsive microRNAs in subtropical maize inbreds.. - MicroRNA signatures of drought signaling in rice root. - Identification of miRNA-mediated drought responsive multi-tiered regulatory network in drought tolerant rice, Nagina 22. - Transcriptome profiling of drought responsive noncoding RNAs and their target genes in rice. - Emerging roles of microRNAs in the mediation of drought stress response in plants. - Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice. - Deregulation of the OsmiR160 target gene OsARF18 causes growth and developmental defects with an alteration of auxin signaling in rice. - OsNAC5 overexpression enlarges root diameter in rice plants leading to enhanced drought tolerance and increased grain yield in the field. - Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. - Identification of drought-responsive microRNAs in tomato using high-throughput sequencing. - Transcriptomic and metabolomic studies disclose key metabolism pathways contributing to well-maintained photosynthesis under the drought and the consequent drought tolerance in rice. - Identification of drought-induced microRNAs in rice. - Genome-wide identification and analysis of drought-responsive microRNAs in Oryza sativa.. - Over-expression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice (Oryza sativa)
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