Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing

Hickory (Carya cathayensis Sarg.) is an economically important woody plant in China, but its long juvenile phase delays yield. MicroRNAs (miRNAs) are critical regulators of genes and important for normal plant development and physiology, including flower development. We used Solexa technology to sequence two small RNA libraries from two floral differentiation stages in hickory to identify miRNAs related to flower development. We identified 39 conserved miRNA sequences from 114 loci belonging to 23 families as well as two novel and ten potential novel miRNAs belonging to nine families. Moreover, 35 conserved miRNA*s and two novel miRNA*s were detected. Twenty miRNA sequences from 49 loci belonging to 11 families were differentially expressed; all were up-regulated at the later stage of flower development in hickory. Quantitative real-time PCR of 12 conserved miRNA sequences, five novel miRNA families, and two novel miRNA*s validated that all were expressed during hickory flower development, and the expression patterns were similar to those detected with Solexa sequencing. Finally, a total of 146 targets of the novel and conserved miRNAs were predicted. This study identified a diverse set of miRNAs that were closely related to hickory flower development and that could help in plant floral induction.     

Identification and characterization of new plant microRNAs using EST analysis

Seventy-five previously known plant microRNAs (miRNAs) were classified into 14 families according to their gene sequence identity. A total of 18,694 plant expressed sequence tags (EST) were found in the GenBank EST databases by comparing all previously known Arabidopsis miRNAs to GenBank‘s plant EST databases with BLAST algorithms. After removing the EST sequences with high numbers (more than 2) of mismatched nucleotides, a total of 812 EST contigs were identified. After predicting and scoring the RNA secondary structure of the 812 EST sequences using mFold software, 338 new potential miRNAs were identified in 60 plant species, miRNAs are widespread. Some microRNAsmay highly conserve in the plant kingdom, and they may have the same ancestor in very early evolution. There is no nucleotide substitution in most miRNAs among many plant species. Some of the new identified potential miRNAs may be induced and regulated by environmental biotic and abiotic stresses. Some may be preferentially expressed in specific tissues, and are regulated by developmental switching. These findings suggest that EST analysis is a good alternative strategy for identifying new miRNA candidates, their targets, and other genes. A large number of miRNAs exist in different plant species and play important roles in plant developmental switching and plant responses to environmental abiotic and biotic stresses as well as signal transduction. Environmental stresses and developmental switching may be the signals for synthesis and regulation of miRNAs in plants. A model for miRNA induction and expression, and gene regulation by miRNA is hypothesized.     

Expression patterns of microRNAs in different organs and developmental stages of a superhybrid rice LYP9 and its parental lines

Heterosis is an important phenomenon, and the molecular mechanisms underlying heterosis are still enigmatic. microRNAs (miRNAs) play vital roles in many aspects of plant development. A set of miRNAs was selected to investigate the roles of miRNAs in heterosis displayed in a superhybrid rice. We analysed the expression patterns of miRNAs in different organs and developmental stages of the superhybrid rice and its parental lines. All possible modes of miRNA action were observed, including additive, high‐ and low‐parent value, above high‐ and below low‐parent value. Different organs and developmental stages exhibited different modes of miRNA expression. Overall, the non‐additive mode is the predominant expression pattern of miRNAs observed in this superhybrid. Many heterotic QTL intervals harbour miRNAs, whose expression patterns reveal their specific roles in different organs and developmental stages. miRNAs regulate the expression levels of target genes that have important functions in plant development. The predominant non‐additive mode of miRNA expression pattern in the hybrid suggests that miRNAs play critical roles in hybrid development, in particular, those miRNAs located in the heterotic QTL intervals may have important roles in heterosis. Our research sheds new light on understanding of the molecular mechanisms of heterosis.     

A Collection of Target Mimics for Comprehensive Analysis of MicroRNA Function in Arabidopsis thaliana

Many targets of plant microRNAs (miRNAs) are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for ∼20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA–resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.     

Identification and characterization of microRNAs and their target genes in Brassica oleracea

The microRNAs are a new class of small non-coding endogenous RNAs with lengths of approximately ~21 nt. MicroRNAs perform their biological function via the degradation of the target mRNAs or by inhibiting protein translation. Until recently, only limited numbers of miRNAs were identified in Brassica oleracea, a vegetable widely cultivated around the world. In present study, 193 potential miRNA candidates were identified from 17 expressed sequence tag (ESTs) and 152 genome survey sequences (GSSs) in B. oleracea. These miRNA candidates were classified into 70 families using a well-defined comparative genome-based computational analysis. Most miRNAs belong to the miRNA169, miR5021, miR156 and miR158 families. Of these, 36 miRNA families are firstly found in Brassica species. Around 1393 B. oleracea genes were predicted as candidate targets of 175 miRNAs. The mutual relationship between miRNAs and the candidate target genes was verified by checking differentially expression levels using quantitative real-time polymerase chain reaction (qRT-PCR) and 5' RLM-RACE analyses. These target genes participate in multiple biological and metabolic processes, including signal transduction, stress response, and plant development. Gene Ontology analysis shows that the 818, 514, and 265 target genes are involved in molecular functions, biological processes, and cellular component respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway enrichment analysis suggests that these miRNAs might regulate 186 metabolic pathways, including those of lipid, energy, starch and sucrose, fatty acid and nitrogen.     

Common functions for diverse small RNAs of land plants

Endogenous small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), are critical components of plant gene regulation. Some abundant miRNAs involved in developmental control are conserved between anciently diverged plants, while many other less-abundant miRNAs appear to have recently emerged in the Arabidopsis thaliana lineage. Using large-scale sequencing of small RNAs, we extended the known diversity of miRNAs in basal plants to include 88 confidently annotated miRNA families in the moss Physcomitrella patens and 44 in the lycopod Selaginella moellendorffii. Cleavage of 29 targets directed by 14 distinct P. patens miRNA families and a trans-acting siRNA (ta-siRNA) was experimentally confirmed. Despite a core set of 12 miRNA families also expressed in angiosperms, weakly expressed and apparently lineage-specific miRNAs accounted for the majority of miRNA diversity in both species. Nevertheless, the molecular functions of several of these lineage-specific small RNAs matched those of angiosperms, despite dissimilarities in the small RNA sequences themselves, including small RNAs that mediated negative feedback regulation of the miRNA pathway and miR390-dependent ta-siRNAs that guided the cleavage of AUXIN RESPONSE FACTOR mRNAs. Diverse, lineage-specific, small RNAs can therefore perform common biological functions in plants.     

Systematic investigation of Amphioxus (Branchiostoma floridae) microRNAs

MicroRNAs (miRNAs) participate in various biological processes via controlling gene activity. Amphioxus is the best available stand-in as the proximate invertebrate ancestor of the vertebrates. Here, we systematically investigated the miRNAs in amphioxus. First, we identified 245 candidate amphioxus miRNAs, in which 183 miRNAs were firstly reported. Second, we gave evidences to support a birth-and-death process of miRNA genes in some families and gave implications for the functional diversification of miRNA during evolution. Third, we identified 47 development-specific expression miRNAs. We found that only 19 miRNAs were expressed in all developmental stages, 16 miRNAs were neurula-specific and 13 miRNAs were larva-specific. In addition, these potential miRNA-targeting genes were mainly classified into development, muscle formation, cell adhesion, and gene regulation categories. Finally, we found 79 immune related genes targeted by 136 miRNAs in amphioxus. In conclusion, our results take an insight into both the function and evolution of the amphioxus miRNAs.