TdERF1, an ethylene response factor associated with dehydration responses in durum wheat (Triticum turgidum L. subsp. durum)

Water deficit and increasing salinization reduce productivity of wheat, the leading crop for human diet. While the complete genome sequence of this crop has not been deciphered, a BAC library screening allowed the isolation of TdERF1, the first ethylene response factor gene from durum wheat. This gene is putatively involved in mediating salt stress tolerance and its characterization provides clues toward understanding the mechanisms underlying the adaptation/tolerance of durum wheat to suboptimal growth conditions. TdERF1 expression is differentially induced by high salt treatment in 2 durum wheat varieties, the salt-tolerant Grecale (GR) and the salt-sensitive Om Rabiaa (OR). To further extend these findings, we show here that the expression of this ERF is correlated with physiological parameters, such as the accumulation of osmo-regulators and membrane integrity, that discriminate between the 2 contrasted wheat genotypes. The data confirm that GR and OR are 2 contrasted wheat genotypes with regard to salt-stress and show that TdERF1 is also induced by water stress with an expression pattern clearly discriminating between the 2 genotypes. These findings suggest that TdERF1 might be involved in responses to salt and water stress providing a potential genetic marker discriminating between tolerant and sensitive wheat varieties.     

Small RNA and degradome deep sequencing reveals important roles of microRNAs in cotton (Gossypium hirsutum L.) response to root-knot nematode Meloidogyne incognita infection

Investigation of cotton response to nematode infection will allow us to better understand the cotton immune defense mechanism and design a better biotechnological approach for efficiently managing pest nematodes in cotton. In this study, we firstly treated cotton by root knot nematode (RKN, Meloidogyne incognita) infections, then we employed the high throughput deep sequencing technology to sequence and genome-widely identify all miRNAs in cotton; finally, we analyzed the functions of these miRNAs in cotton response to RKN infections. A total of 266 miRNAs, including 193 known and 73 novel miRNAs, were identified by deep sequencing technology, which belong to 67 conserved and 66 novel miRNA families, respectively. A majority of identified miRNA families only contain one miRNA; however, miR482 family contains 14 members and some others contain 2–13 members. Certain miRNAs were specifically expressed in RKN-infected cotton roots and others were completely inhibited by RKN infection. A total of 50 miRNAs were differentially expressed after RKN infection, in which 28 miRNAs were up-regulated and 22 were inhibited by RKN treatment. Based on degradome sequencing, 87 gene targets were identified to be targeted by 57 miRNAs. These miRNA-targeted genes are involved in the interaction of cotton plants and nematode infection. Based on GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, 466 genes from all 636 miRNA targets were mapped to 6340 GO terms, 181 genes from 228 targets of differentially expressed miRNAs were mapped to 1588 GO terms. The GO terms were then categorized into the three main GO classes: biological processes, cellular components, and molecular functions. The targets of differentially expressed miRNAs were enriched in 43 GO terms, including 22 biological processes, 10 cellular components, and 11 molecular functions (p < 0.05). Many identified processes were associated with organism responses to the environmental stresses, including regulation of nematode larval development, response to nematode, and response to flooding. Our results will enhance the study and application of developing new cotton cultivars for nematode resistance.     

Transposable element-associated microRNA hairpins produce 21-nt sRNAs integrated into typical microRNA pathways in rice

microRNAs (miRNAs) are a class of small RNAs (sRNAs) of ～21 nucleotides (nt) in length processed from foldback hairpins by DICER-LIKE1 (DCL1) or DCL4. They regulate the expression of target mRNAs by base pairing through RNA-induced silencing complex (RISC). In the RISC, ARGONAUTE1 (AGO1) is the key protein that cleaves miRNA targets at position ten of a miRNA:target duplex. The authenticity of many annotated rice miRNA hairpins is under debate because of their homology to repeat sequences. Some of them, like miR1884b, have been removed from the current release of miRBase based on incomplete information. In this study, we investigated the association of transposable element (TE)-derived miRNAs with typical miRNA pathways (DCL1/4- and AGO1-dependent) using publicly available deep sequencing datasets. Seven miRNA hairpins with 13 unique sRNAs were specifically enriched in AGO1 immunoprecipitation samples and relatively reduced in DCL1/4 knockdown genotypes. Interestingly, these species are ～21-nt long, instead of 24-nt as annotated in miRBase and the literature. Their expression profiles meet current criteria for functional annotation of miRNAs. In addition, diagnostic cleavage tags were found in degradome datasets for predicted target mRNAs. Most of these miRNA hairpins share significant homology with miniature inverted-repeat transposable elements, one type of abundant DNA transposons in rice. Finally, the root-specific production of a 24-nt miRNA-like sRNA was confirmed by RNA blot for a novel EST that maps to the 3′-UTR of a candidate pseudogene showing extensive sequence homology to miR1884b hairpin. Our data are consistent with the hypothesis that TEs can serve as a driving force for the evolution of some MIRNAs, where co-opting of DICER-LIKE1/4 processing and integration into AGO1 could exapt transcribed TE-associated hairpins into typical miRNA pathways.     

Deep sequencing identified potential miRNAs involved in defence response,stress and plant growth characteristics of wild genotypes of cardamom

• Cardamom has long been used as a food flavouring agent and in ayurvedic medicines for mouth ulcers, digestive problems and even depression. Extensive occurrence of pests and diseases adversely affect its cultivation and result in substantial reductions in total production and productivity. Numerous studies revealed the significant role of miRNAs in plant biotic stress responses.
• In the current study, miRNA profiling of cultivar and wild cardamom genotypes was performed using an Ion Proton sequencer.
• We identified 161 potential miRNAs representing 42 families, including monocot/tissue‐specific and 14 novel miRNAs in both genotypes. Significant differences in miRNA family abundance between the libraries were observed in read frequencies. A total of 19 miRNAs (from known miRNAs) displayed a twofold difference in expression between wild and cultivar genotypes. We found 1168 unique potential targets for 40 known miRNA families in wild and 1025 potential targets for 42 known miRNA families in cultivar genotypes. The differential expression analysis revealed that most miRNAs identified were highly expressed in cultivars and, furthermore, lower expression of miR169 and higher expression of miR529 in wild cardamom proved evidence that wild genotypes have stronger drought stress tolerance and floral development than cultivars.
• Potential targets predicted for the newly identified miRNAs from the miRNA libraries of wild and cultivar cardamom genotypes involved in metabolic and developmental processes and in response to various stimuli. qRT‐PCR confirmed miRNAs were differentially expressed between wild and cultivar genotypes. Furthermore, four target genes were validated experimentally to confirm miRNA–mRNA target pairing using RNA ligase‐mediated 5′ Rapid Amplification of cDNA Ends (5′RLM‐RACE) PCR.

     

Identification of drought-responsive microRNAs in tomato using high-throughput sequencing

Drought is a major abiotic stress affecting crop productivity and quality. As a class of noncoding RNA, microRNA (miRNA) plays important roles in plant growth, development, and stress response. However, their response and roles in tomato drought stress is largely unknown. Here, by using high-throughput sequencing, we compared the miRNA profiles before and after drought treatment in two tomato genotypes: M82, a drought-sensitive cultivated tomato (Solanum lycopersicum), and IL2-5, a drought-tolerant introgression line derived from M82 and the tomato wild species S. pennellii (LA0716). A total of 108 conserved and 208 novel miRNAs were identified, among them, 32 and 68 were significantly changed in expression after stress. Further, 1936 putative target genes were predicted for those differentially-expressed miRNAs. Gene ontology and pathway analysis showed that many of the target genes were involved in stress resistance, such as genes in GO terms including response to stress, defense response, response to stimulus, phosphorylation, and signal transduction. Our results suggested that miRNAs play an essential role in the drought response of tomato. This work will help to further characterize specific miRNAs functioning in drought tolerance.     

An integrative approach to identify hexaploid wheat miRNAome associated with development and tolerance to abiotic stress

Background

Wheat is a major staple crop with broad adaptability to a wide range of environmental conditions. This adaptability involves several stress and developmentally responsive genes, in which microRNAs (miRNAs) have emerged as important regulatory factors. However, the currently used approaches to identify miRNAs in this polyploid complex system focus on conserved and highly expressed miRNAs avoiding regularly those that are often lineage-specific, condition-specific, or appeared recently in evolution. In addition, many environmental and biological factors affecting miRNA expression were not yet considered, resulting still in an incomplete repertoire of wheat miRNAs.

Results

We developed a conservation-independent technique based on an integrative approach that combines machine learning, bioinformatic tools, biological insights of known miRNA expression profiles and universal criteria of plant miRNAs to identify miRNAs with more confidence. The developed pipeline can potentially identify novel wheat miRNAs that share features common to several species or that are species specific or clade specific. It allowed the discovery of 199 miRNA candidates associated with different abiotic stresses and development stages. We also highlight from the raw data 267 miRNAs conserved with 43 miRBase families. The predicted miRNAs are highly associated with abiotic stress responses, tolerance and development. GO enrichment analysis showed that they may play biological and physiological roles associated with cold, salt and aluminum (Al) through auxin signaling pathways, regulation of gene expression, ubiquitination, transport, carbohydrates, gibberellins, lipid, glutathione and secondary metabolism, photosynthesis, as well as floral transition and flowering.

Conclusion

This approach provides a broad repertoire of hexaploid wheat miRNAs associated with abiotic stress responses, tolerance and development. These valuable resources of expressed wheat miRNAs will help in elucidating the regulatory mechanisms involved in freezing and Al responses and tolerance mechanisms as well as for development and flowering. In the long term, it may help in breeding stress tolerant plants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1490-8) contains supplementary material, which is available to authorized users.     

Circulating microRNA signature of steroid‐induced osteonecrosis of the femoral head

Objectives

Steroid‐induced osteonecrosis of the femoral head (ONFH) is a common orthopaedic disease of which early detection remains clinically challenging. Accumulating evidences indicated that circulating microRNAs (miRNAs) plays vital roles in the development of several bone diseases. However, the association between circulating miRNAs and steroid‐induced ONFH remains elusive.

Materials and methods

miRNA microarray was performed to identify the differentially abundant miRNAs in the serums of systemic lupus erythematosus (SLE) patients with steroid‐induced ONFH as compared with SLE control and healthy control group. We predicted the potential functions of these differentially abundant miRNAs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and reconstructed the regulatory networks of miRNA‐mRNA interactions.

Results

Our data indicated that there were 11 differentially abundant miRNAs (2 upregulated and 9 downregulated) between SLE‐ONFH group and healthy control group and 42 differentially abundant miRNAs (14 upregulated and 28 downregulated) between SLE‐ONFH group and SLE control group. We also predicted the potential functions of these differentially abundant miRNAs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and reconstructed the regulatory networks of miRNA‐mRNA interactions.

Conclusions

These findings corroborated the idea that circulating miRNAs play significant roles in the development of ONFH and may serve as diagnostic markers and therapeutic targets.     

Identification and comparative analysis of aluminum-induced microRNAs conferring plant tolerance to aluminum stress in soybean

Aluminum (Al) toxicity in acidic soils is a major factor restricting crop production. Although the molecular mechanisms of Al responses have been extensively investigated, microRNA (miRNA) mediated differential Al tolerance in different soybean genotypes remains largely unknown. In this study, two soybean [Glycine max (L.) Merr.] genotypes, Al-tolerant BX10 and Al-sensitive BD2, were treated with 0 and 50 μM AlCl3 and then used to construct the miRNA libraries for deep sequencing. Results revealed 453 miRNAs, whose expression patterns were affected by Al stress. We also identified 32 differentially expressed miRNAs: 19 in BX10, 7 in BD2, and 6 in both genotypes. The gene ontology analysis of their putative target genes indicated that stress-responsive genes and amino-acid-metabolism-related processes preferentially existed in BX10. Comprehensive analysis demonstrated that conserved miRNAs, such as gma-miR166k/o, gma-miR390g, and gma-miR396c/k, mediated root elongation in BX10, whereas gma-miR169r triggered oxidative stress in BD2. These processes could be regarded as important mechanisms conferring differential Al tolerance in BX10 and BD2. This study provided new insights into different Al response mechanisms in various soybean genotypes.