Microarray-based screening of the microRNAs associated with caryopsis development in Oryza sativa

Plant microRNAs modulate diverse developmental processes by regulating expression of their target genes. To explore potential miRNA-guided gene regulation in developing rice (Oryza sativa L.) caryopses, a miRNA microarray was used to identify miRNAs present at the different developmental stages. We found that 27 miRNAs, of which 16 were conserved miRNAs, were present in developing caryopses. High expression levels were detected for miR159, miR167, and miR530 at the morphogenesis stage and for miR169, miR435, and miR528 at the stage of accumulation of metabolites. Next, 26 target genes were predicted for seven of the detected miRNAs and the expression profiles of these miRNAs and their corresponding target genes were examined in developing caryopses. Our results suggest that the miRNAs and their target genes examined at the two distinct stages could contribute to the developmental progress of rice caryopses in concert with phytohormone signalling.     

Identification and expression analysis of miRNAs in germination and seedling growth of Tibetan hulless barley

Germination and seedling growth are crucial for plant development and agricultural production. While, the regulatory mechanisms during these processes in Tibetan hulless barley (Hordeum vulgare L. var. nudum) are not well understood. Given the regulatory roles of microRNAs (miRNAs) in crop plants and the irreplaceability of barley in the highland area of China, we herein presented a genome-wide survey of miRNAs to reveal a potential regulatory network in the early developmental stages of two Tibetan hulless barleys, from which a total of 156 miRNAs was identified including 35 known and 121 novel ones. Six of the identified novel miRNAs were further experimentally validated. According to the evolutionary analysis, miR156, miR166, miR168, and miR171 were conserved across Tibetan hulless barleys and eight other seed plants. Expression profiles of ten known miRNAs showed that they were involved in phytohormone signaling, carbohydrate and lipid metabolism, as well as juvenile-adult transition during barley development. Moreover, a total of 1280 genes targeted by 101 miRNAs were predicted from both barley libraries. Three genes (PLN03212, MATE eukaryotic, and GRAS) were validated via the RNA ligase-mediated 5′-rapid amplification of cDNA ends (RLM-5' RACE) to be the targets of hvu-miR159a, hvu-miR166a, and hvu-miR171-3p, respectively. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of putative targets, the most abundant pathways were related to “metabolism”.These results revealed that miRNA-target pairs participating in the regulation of multigene expression and the embryonic development of Tibetan hulless barleys were controlled by complex mechanisms involving the concordant expression of different miRNAs and feedback loops among miRNAs as well as their targets. The study provides insight into the regulatory network of barley miRNAs for better understanding of miRNA functions during germination and seedling growth.     

The Regulatory Roles of microRNAs and Associated Target Genes during Early Somatic Embryogenesis in <i>Liriodendron Sino-Americanum</i>

Somatic cells respond to considerable stress, and go through a series of phytohormone pathways, then forming an embryo. The developmental process is recorded as somatic embryogenesis (SE). One of the key components regulating SE are the microRNAs (miRNAs). Despite previous studies, it is still not clear exactly how miRNAs exert their function of regulating targets during conditionally activated early SE. Here, we use Liriodendron sino-americanum as a model system and perform a combined analysis of microfluidic chips and degradome sequencing to study this process. We identified a total of 386 conserved miRNAs and 153 novel miRNAs during early SE. According to the ANOVA test, 239 miRNAs showed 12 distinct expression patterns. Through degradome sequencing, 419 targets and 198 targets were identified for 136 known miRNAs and 37 novel miRNAs, respectively. Gene Ontology (GO) and metabolism pathway enrichment analysis revealed that these targets were significantly involved in oxidation-reduction processes, calmodulin-mediated signal transduction pathways and carbohydrate metabolism. The genes that were related to stress responses, phytohormone pathways and plant metabolism were identified within the targets of miR319, miR395, miR408, miR472, miR482, miR390, miR2055, miR156, miR157, miR171, miR396, miR397, miR529, miR535 and miR159. According to promoter analysis, various cis-acting elements related to plant growth and development, phytohormones response and stress response were present in the promoter of the miRNAs. The differential expression patterns of 11 miRNA-target modules were confirmed by real-time quantitative PCR. The study demonstrated that the miRNA plays an important role in the early SE process by regulating its target and then participating in carbohydrate metabolism and stress response. It also provided a valuable resource for further research in determining the genetic mechanism of SE, and then facilitating breeding programs on plants.     

Identification of miRNAs and Their Target Genes in Peach (Prunus persica L.) Using High-Throughput Sequencing and Degradome Analysis

MicroRNAs play critical roles in various biological and metabolic processes. The function of miRNAs has been widely studied in model plants such as Arabidopsis and rice. However, the number of identified miRNAs and related miRNA targets in peach (Prunus persica) is limited. To understand further the relationship between miRNAs and their target genes during tissue development in peach, a small RNA library and three degradome libraries were constructed from three tissues for deep sequencing. We identified 117 conserved miRNAs and 186 novel miRNA candidates in peach by deep sequencing and 19 conserved miRNAs and 13 novel miRNAs were further evaluated for their expression by RT-qPCR. The number of gene targets that were identified for 26 conserved miRNA families and 38 novel miRNA candidates, were 172 and 87, respectively. Some of the identified miRNA targets were abundantly represented as conserved miRNA targets in plant. However, some of them were first identified and showed important roles in peach development. Our study provides information concerning the regulatory network of miRNAs in peach and advances our understanding of miRNA functions during tissue development.     

Identification and validation of Asteraceae miRNAs by the expressed sequence tag analysis

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a vital role in the regulation of gene expression. Despite their identification in hundreds of plant species, few miRNAs have been identified in the Asteraceae, a large family that comprises approximately one tenth of all flowering plants. In this study, we used the expressed sequence tag (EST) analysis to identify potential conserved miRNAs and their putative target genes in the Asteraceae. We applied quantitative Real-Time PCR (qRT-PCR) to confirm the expression of eight potential miRNAs in Carthamus tinctorius and Helianthus annuus. We also performed qRT-PCR analysis to investigate the differential expression pattern of five newly identified miRNAs during five different cotyledon growth stages in safflower. Using these methods, we successfully identified and characterized 151 potentially conserved miRNAs, belonging to 26 miRNA families, in 11 genus of Asteraceae. EST analysis predicted that the newly identified conserved Asteraceae miRNAs target 130 total protein-coding ESTs in sunflower and safflower, as well as 433 additional target genes in other plant species. We experimentally confirmed the existence of seven predicted miRNAs, (miR156, miR159, miR160, miR162, miR166, miR396, and miR398) in safflower and sunflower seedlings. We also observed that five out of eight miRNAs are differentially expressed during cotyledon development. Our results indicate that miRNAs may be involved in the regulation of gene expression during seed germination and the formation of the cotyledons in the Asteraceae. The findings of this study might ultimately help in the understanding of miRNA-mediated gene regulation in important crop species.     

Identification and characterization of salt-responsive microRNAs in Populus tomentosa by high-throughput sequencing

Salt is one of the main environmental factors limiting plant growth and a better understanding of mechanisms of salt stress would aid efforts to bolster plant salt tolerance. MicroRNAs are well known for their important regulatory roles in response to abiotic stress in plants. In this study, high-throughput sequencing was employed to identify miRNAs in Populus tomentosa plantlets treated or not with salt (200 mM for 10 h). We found 141 conserved miRNAs belonging to 31 families, 29 non-conserved but previously-known miRNAs belonging to 26 families, and 17 novel miRNAs. Under salt stress, 19 miRNAs belonging to seven conserved miRNA families were significantly downregulated, and two miRNAs belonging to two conserved miRNA families were upregulated. Of seven non-conserved miRNAs with significantly altered expression, five were downregulated and two were upregulated. Furthermore, eight miRNAs were validated by qRT-PCR and their dynamic differential expressions were analyzed. In addition, 269 target genes of identified miRNAs were predicted and categorized by function. These results provide new insights into salt-responsive miRNAs in Populus.