Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis

Flowering is the primary trait affected by ambient temperature changes. Plant microRNAs (miRNAs) are small non-coding RNAs playing an important regulatory role in plant development. In this study, to elucidate the mechanism of flowering-time regulation by small RNAs, we identified six ambient temperature-responsive miRNAs (miR156, miR163, miR169, miR172, miR398 and miR399) in Arabidopsis via miRNA microarray and northern hybridization analyses. We also determined the expression profile of 120 unique miRNA loci in response to ambient temperature changes by miRNA northern hybridization analysis. The expression of the ambient temperature-responsive miRNAs and their target genes was largely anticorrelated at two different temperatures (16 and 23°C). Interestingly, a lesion in short vegetative phase (SVP), a key regulator within the thermosensory pathway, caused alteration in the expression of miR172 and a subset of its target genes, providing a link between a thermosensory pathway gene and miR172. The miR172-overexpressing plants showed a temperature-independent early flowering phenotype, suggesting that modulation of miR172 expression leads to temperature insensitivity. Taken together, our results suggest a genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs under non-stress temperature conditions.     

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.     

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.     

Analyses of a Glycine max Degradome Library Identify microRNA Targets and MicroRNAs that Trigger Secondary SiRNA Biogenesis

Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs. In this study, a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced. 428 potential targets of small interfering RNAs and 25 novel miRNA families were identified. A total of 211 potential miRNA targets, including 174 conserved miRNA targets and 37 soybean-specific miRNA targets, were identified. Among them, 121 targets were first discovered in soybean. The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing. The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean, and nine Auxin Response Factors were identified as TAS3 siRNA targets. Twenty-three miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean. These targets were guided by five miRNAs: gma-miR393, gma-miR1508, gma-miR1510, gma-miR1514, and novel-11. Multiple targets of these secondary siRNAs were detected. These 23 miRNA targets may be the putative novel TAS genes in soybean. Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.     

Identification and Characterization of ABA-Responsive MicroRNAs in Rice

MicroRNAs(miRNAs) are endogenous non-coding small RNAs that silence genes through mRNA degradation or translational inhibition.The phytohormone abscisic acid(ABA) is essential for plant development and adaptation to abiotic and biotic stresses.In Arabidopsis,miRNAs are implicated in ABA functions.However,ABA-responsive miRNAs have not been systematically studied in rice.Here high throughput sequencing of small RNAs revealed that 107 miRNAs were differentially expressed in the rice ABA deficient mutant,Osabal.Of these,13 were confirmed by stem-loop RT-PCR.Among them,miR1425-5P,miR169 a,miR169n,miR390-5P,miR397 a and miR397 b were up-regulated,but miR162 b reduced in expression in Osabal.The targets of these 13 miRNAs were predicted and validated by gene expression profiling.Interestingly,the expression levels of these miRNAs and their targets were regulated by ABA.Cleavage sites were detected on 7 of the miRNA targets by 5'-Rapid Amplification of cDNA Ends(5'-RACE).Finally,miR162 b and its target OsTREl were shown to affect rice resistance to drought stress,suggesting that miR162 b increases resistance to drought by targeting OsTREl.Our work provides important information for further characterization and functional analysis of ABA-responsive miRNAs in rice.     

Conservation and divergence in plant microRNAs

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression in eukaryotic cells. The past decade has seen an explosion in our understanding of the sets of miRNA genes encoded in the genomes in different species of plants and the mechanisms by which miRNAs interact with target RNAs. A subset of miRNA families (and their binding sites in target RNAs) are conserved between angiosperms and basal plants, suggesting they predate the divergence of existing lineages of plants. However, the majority of miRNA families expressed by any given plant species have a narrow phylogenetic distribution. As a group, these "young" miRNAs genes appear to be evolutionarily fluid and lack clearly understood biological function. The goal of this review is to summarize our understanding of the sets of miRNA genes and miRNA targets that exist in various plant species and to discuss hypotheses that explain the patterns of conservation and divergence observed among microRNAs in plants.     

Comparative expression of miRNA genes and miRNA-based AFLP marker analysis in cultivated tetraploid cottons

MicroRNAs (miRNAs) are a class of small non-coding RNAs that down-regulate gene expression in a sequence specific manner to control plant growth and development. The identification and characterization of miRNAs are critical steps in finding their target genes and elucidating their functions. The objective of the present study was to assess the genetic variation of miRNA genes through expression comparisons and miRNA-based AFLP marker analysis. Seven miRNAs were first selected for RT-PCR and four for quantitative RT-PCR analysis that showed considerably high or differential expression levels in early stages of boll development. Except for miR160a, differential gene expression of miR171, 390a, and 396a was detected in early developing bolls at one or more timepoints between two cultivated cotton cultivars, Pima Phy 76 (Gossypium barbadense) and Acala 1517-99 (Gossypium hirsutum). Our further work demonstrated that genetic diversity of miRNA genes can be assessed by miRNA-AFLP analysis using primers designed from 22 conserved miRNA genes in combination with AFLP primers. Homologous miRNA genes can be also identified and isolated for sequencing and confirmation using this homology-based genotyping approach. This strategy offers an alternative to isolating a full length of miRNA genes and their up-stream and down-stream sequences. The significance of the expression and sequence differences of miRNAs between cotton species or genotypes needs further studies.