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.     

Identification of reference microRNAs and suitability of archived hemopoietic samples for robust microRNA expression profiling

In many cancers, including neuroblastoma, microRNA (miRNA) expression profiling of peripheral blood (PB) and bone marrow (BM) may increase understanding of the metastatic process and lead to the identification of clinically informative biomarkers. The quality of miRNAs in PB and BM samples archived in PAXgene? blood RNA tubes from large-scale clinical studies and the identity of reference miRNAs for standard reporting of data are to date unknown. In this study, we evaluated the reliability of expression profiling of 377 miRNAs using quantitative polymerase chain reaction (qPCR) in PB and BM samples (n = 90) stored at ?80 °C for up to 5 years in PAXgene? blood RNA tubes. There was no correlation with storage time and variation of expression for any single miRNA (r < 0.50). The profile of miRNAs isolated as small RNAs or co-isolated with small/large RNAs was highly correlated (r = 0.96). The mean expression of all miRNAs and the geNorm program identified miR-26a, miR-28-5p, and miR-24 as the most stable reference miRNAs. This study describes detailed methodologies for reliable miRNA isolation and profiling of PB and BM, including reference miRNAs for qPCR normalization, and demonstrates the suitability of clinical samples archived at ?80 °C into PAXgene? blood RNA tubes for miRNA expression studies.     

MicroRNA–target gene responses to lead-induced stress in cotton (Gossypium hirsutum L.)

MicroRNAs (miRNAs) play key roles in plant responses to various metal stresses. To investigate the miRNA-mediated plant response to heavy metals, cotton (Gossypium hirsutum L.), the most important fiber crop in the world, was exposed to different concentrations (0, 25, 50, 100, and 200 µM) of lead (Pb) and then the toxicological effects were investigated. The expression patterns of 16 stress-responsive miRNAs and 10 target genes were monitored in cotton leaves and roots by quantitative real-time PCR (qRT-PCR); of these selected genes, several miRNAs and their target genes are involved in root development. The results show a reciprocal regulation of cotton response to lead stress by miRNAs. The characterization of the miRNAs and the associated target genes in response to lead exposure would help in defining the potential roles of miRNAs in plant adaptation to heavy metal stress and further understanding miRNA regulation in response to abiotic stress.     

Genome-wide identification and expression analysis of heat-responsive and novel microRNAs in Populus tomentosa

Plant microRNAs have a vital role in various abiotic stress responses by regulating gene expression. Heat stress is one of the most severe abiotic stresses, and affects plant growth and development, even leading to death. To identify heat-responsive miRNAs at the genome-wide level in Populus, Solexa sequencing was employed to sequence two libraries from Populus tomentosa, treated and untreated by heat stress. Sequence analysis identified 134 conserved miRNAs belonging to 30 miRNA families, and 16 novel miRNAs belonging to 14 families. Among these miRNAs, 52 miRNAs from 15 families were responsive to heat stress and most of them were down-regulated. qRT-PCR analysis confirmed that the conserved and novel miRNAs were expressed in P. tomentosa, and revealed similar expression trends to the Solexa sequencing results obtained under heat stress. One hundred and nine targets of the novel miRNAs were predicted. This study opens up a new avenue for understanding the regulatory mechanisms of miRNAs involvement in the heat stress response of trees.     

Genome-wide profiling of novel and conserved <Emphasis Type="Italic">Populus</Emphasis> microRNAs involved in pathogen stress response by deep sequencing

MicroRNAs (miRNAs) are small RNAs, generally of 20–23 nt, that down-regulate target gene expression during development, differentiation, growth, and metabolism. In Populus, extensive studies of miRNAs involved in cold, heat, dehydration, salinity, and mechanical stresses have been performed; however, there are few reports profiling the miRNA expression patterns during pathogen stress. We obtained almost 38 million raw reads through Solexa sequencing of two libraries from Populus inoculated and uninoculated with canker disease pathogen. Sequence analyses identified 74 conserved miRNA sequences belonging to 37 miRNA families from 154 loci in the Populus genome and 27 novel miRNA sequences from 35 loci, including their complementary miRNA* strands. Intriguingly, the miRNA* of three conserved miRNAs were more abundant than their corresponding miRNAs. The overall expression levels of conserved miRNAs increased when subjected to pathogen stress, and expression levels of 33 miRNA sequences markedly changed. The expression trends determined by sequencing and by qRT-PCR were similar. Finally, nine target genes for three conserved miRNAs and 63 target genes for novel miRNAs were predicted using computational analysis, and their functions were annotated. Deep sequencing provides an opportunity to identify pathogen-regulated miRNAs in trees, which will help in understanding the regulatory mechanisms of plant defense responses during pathogen infection.     

Identification of the Conserved and Novel miRNAs in Mulberry by High-Throughput Sequencing

miRNAs are a class of non-coding endogenous small RNAs. They play vital roles in plant growth, development, and response to biotic and abiotic stress by negatively regulating genes. Mulberry trees are economically important species with multiple uses. However, to date, little is known about mulberry miRNAs and their target genes. In the present study, three small mulberry RNA libraries were constructed and sequenced using high-throughput sequencing technology. Results showed 85 conserved miRNAs belonging to 31 miRNA families and 262 novel miRNAs at 371 loci. Quantitative real-time PCR (qRT-PCR) analysis confirmed the expression pattern of 9 conserved and 5 novel miRNAs in leaves, bark, and male flowers. A total of 332 potential target genes were predicted to be associated with these 113 novel miRNAs. These results provide a basis for further understanding of mulberry miRNAs and the biological processes in which they are involved.     

Expression profiles of precursor and mature microRNAs under dehydration and high salinity shock in Populus euphratica

MicroRNAs (miRNAs) are small non-coding RNAs that play vital roles in plant abiotic stress responses via cleavage or translational inhibition of their target mRNAs. Populus euphratica is a typical stress-resistant sessile organism that grows in desert areas. Here, we identified sequences of 12 miRNA precursors from 11 families and 13 mature miRNAs from 12 families by PCR amplification in P. euphratica. To detect expression differences in mature miRNAs and their precursors under dehydration and high salinity shock in P. euphratica, we examined 14 miRNA precursors from 13 miRNA families and 17 mature miRNAs from 17 miRNA families using the SYBR Green RT–PCR assay. This is the first report of expression profiles for both precursor and mature miRNAs in P. euphratica. By profiling both the mature miRNAs and the precursors under abiotic stress shock, it was possible to identify miRNA whose processing is regulated during stress shock environments. A majority of the genes predicted to be targets for plant miRNAs are involved in development, stress resistance and metabolic processes. We have cloned and experimentally identified in vivo five of the predicted target genes and quantified the five target mRNAs from the same RNA sample simultaneously. Based on this study, we propose some regulatory pathways that illustrate the important role that miRNAs play in response to abiotic stress shock in P. euphratica.     

Identification of MicroRNAs and Transcript Targets in Camelina sativa by Deep Sequencing and Computational Methods

Camelina sativa is an annual oilseed crop that is under intensive development for renewable resources of biofuels and industrial oils. MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play key roles in diverse plant biological processes. Here, we conducted deep sequencing on small RNA libraries prepared from camelina leaves, flower buds and two stages of developing seeds corresponding to initial and peak storage products accumulation. Computational analyses identified 207 known miRNAs belonging to 63 families, as well as 5 novel miRNAs. These miRNAs, especially members of the miRNA families, varied greatly in different tissues and developmental stages. The predicted miRNA target genes are involved in a broad range of physiological functions including lipid metabolism. This report is the first step toward elucidating roles of miRNAs in C. sativa and will provide additional tools to improve this oilseed crop for biofuels and biomaterials.     

Genome-Wide Identification and Analysis of MicroRNAs Involved in Witches’-Broom Phytoplasma Response in Ziziphus jujuba

MicroRNAs (miRNAs) play an important role in responding to biotic and abiotic stresses in plants. Jujube witches’-broom a phytoplasma disease of Ziziphus jujuba is prevalent in China and is a serious problem to the industry. However, the molecular mechanism of the disease is poorly understood. In this study, genome-wide identification and analysis of microRNAs in response to witches’-broom was performed. A total of 85 conserved miRNA unique sequences belonging to 32 miRNA families and 24 novel miRNA unique sequences, including their complementary miRNA* strands were identified from small RNA libraries derived from a uninfected and witches’-broom infected Z. jujuba plant. Differentially expressed miRNAs associated with Jujube witches’-broom disease were investigated between the two libraries, and 12 up-regulated miRNAs and 10 down- regulated miRNAs identified with more than 2 fold changes. Additionally, 40 target genes of 85 conserved miRNAs and 49 target genes of 24 novel miRNAs were predicted and their putative functions assigned. Using the modified 5’-RACE method, we confirmed that SPL and MYB were cleaved by miR156 and miR159, respectively. Our results provide insight into the molecular mechanisms of witches’-broom disease in Z. jujuba.