System analysis of microRNAs in the development and aluminium stress responses of the maize root system

MicroRNAs (miRNAs) are a class of regulatory small RNAs (sRNAs) that down‐regulate target genes through mRNA cleavage or translational inhibition. miRNA is known to play an important role in the root development and environmental responses in both the Arabidopsis and rice. However, little information is available to form a complete view of miRNAs in the development of the maize root system and Al stress responses in maize. Four sRNA libraries were generated and sequenced from the early developmental stage of primary roots (PRY), the later developmental stage of maize primary roots (PRO), seminal roots (SR) and crown roots (CR). Through integrative analysis, we identified 278 miRNAs (246 conserved and 32 novel ones) and found that the expression patterns of miRNAs differed dramatically in different maize roots. The potential targets of the identified conserved and novel miRNAs were also predicted. In addition, our data showed that CR is more resistant to Al stress compared with PR and SR, and the differentially expressed miRNAs are likely to play significant roles in different roots in response to environmental stress such as Al stress. Here, we demonstrate that the expression patterns of miRNAs are highly diversified in different maize roots. The differentially expressed miRNAs are correlated with both the development and environmental responses in the maize root. This study not only improves our knowledge about the roles of miRNAs in maize root development but also reveals the potential role of miRNAs in the environmental responses of different maize roots.     

Genome-wide identification of cold-responsive and new microRNAs in Populus tomentosa by high-throughput sequencing

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate the expression of target mRNAs in plant growth, development, abiotic stress responses, and pathogen responses. Cold stress is one of the most common abiotic factors affecting plants, and it adversely affects plant growth, development, and spatial distribution. To understand the roles of miRNAs under cold stress in Populus tomentosa, we constructed two small RNA libraries from plantlets treated or not with cold conditions (4 °C for 8 h). High-throughput sequencing of the two libraries identified 144 conserved miRNAs belonging to 33 miRNA families and 29 new miRNAs (as well as their corresponding miRNA¹s) belonging to 23 miRNA families. Differential expression analysis showed that 21 miRNAs were down-regulated and nine miRNAs were up-regulated in response to cold stress. Among them, 19 cold-responsive miRNAs, two new miRNAs and their corresponding miRNA¹s were validated by qRT-PCR. A total of 101 target genes of the new miRNAs were predicted using a bioinformatics approach. These target genes are involved in growth and resistance to various stresses. The results demonstrated that Populus miRNAs play critical roles in the cold stress response.     

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.     

Role of let-7b/Fzd4 axis in mitochondrial biogenesis through wnt signaling: In neonatal and adult megakaryocytes

BackgroundMegakaryocytes (MKs), a rare population of bone marrow cells, are responsible for the production of platelets. Sick neonates are predisposed to developing thrombocytopenia (platelet count <150 × 109/L) and neonates are affected by several megakaryocyte disorders as compared to adults.HypothesisMicroRNAs (miRNAs) have been shown to crucially involve in the regulation of stem-cell differentiation in normal as well as malignant hematopoiesis, but their role in regulation of biological differences between adult and neonatal megakaryopoiesis is unknown.MethodsTo study this, we cultured human cord blood (CB) and peripheral blood (PB) derived CD34⁺ cells in the presence of thrombopoietin for 14 days and collected cultures expressing >90% CD41⁺ by flow cytometry and studied 88 miRNAs involved in stem cell development and differentiation. miRNA validation studies were performed in Dami cell line.ResultsOut of 88 miRNAs involved in stem cell development, let-7b was the only miRNA down regulated (∼10-fold) in neonates compared to adult-MKs. Let-7b has not been previously described in MKs, however reduced expression of let-7b was found in several human cancers, suggesting that it functions as a tumor suppressor. Our results showed the inhibitory effect of let-7b on wnt signaling pathway by regulating Fzd4 (frizzled family receptor 4) and thereby regulating proliferation as well as differentiation. Let-7b down regulation induced mitochondrial biogenesis and its markers PGC-1α and NRF1 during megakaryocyte development.ConclusionsOur findings for the first time unveil the novel role of let-7b/Fzd4 axis through wnt signaling by regulating mitochondrial biogenesis during megakaryocyte development.     

Silicon mitigates cadmium inhibitory effects in young maize plants

The influence of silicon on the growth of maize plants cultivated in hydroponics in the presence of cadmium (5 μM) was investigated. Four different treatments were used: Control (C), Cadmium (Cd), Silicon (Si) and Cadmium plus Silicon (Cd + Si). The Si concentration was 35 mM. Thirteen-day-old plants were harvested. Growth parameters (length of primary seminal root, leaf area of first and second fully developed leaves, fresh and dry weight of below- and above-ground parts of the plants), and Cd concentration and total amount of Cd in the below- and above-ground parts were determined. In roots, the development of the endodermal barrier was observed by fluorescent staining with Fluorol yellow 088.Inhibitory effects of Cd on plant growth were observed. Silicon treatment in the absence of Cd had positive effects on most of observed growth parameters compared with the control. Moreover, Si in the Cd + Si treatment improved all growth parameters compared with the cadmium treatment. Silicon increased the cell-wall extensibility both in Si and Cd + Si treatments when compared with the control. Alleviation of the Cd-inhibitory effect on maize plants by Si was not due to exclusion of Cd from the plant; in contrast, Cd concentration in below- and above-ground plant parts and the total amount of Cd per plant were significantly higher in the Cd + Si plants than in the Cd treatment. The increased Cd content in Cd + Si plants was correlated with the development of the endodermis; during the second stage of endodermal development, suberin lamellae were formed at a greater distance from the root apex in the Cd + Si than in the Cd treatment. Silicon itself did not influence the development of suberin lamellae in the maize roots compared with the control.     

MicroRNA miR-7 is preferentially expressed in endocrine cells of the developing and adult human pancreas

MicroRNAs (miRNA) are small non-coding RNAs that inhibit gene expression through binding to complementary messenger RNA sequences. miRNAs have been predicted to target genes important for pancreas development, proper endocrine cell function and metabolism. We previously described that miRNA-7 (miR-7) was the most abundant and differentially expressed islet miRNA, with 200-fold higher expression in mature human islets than in acinar tissue. Here we have analyzed the temporal and spatial expression of miR-7 in human fetal pancreas from 8 to 22 weeks of gestational age (wga). Human fetal (8–22 wga) and adult pancreases were processed for immunohistochemistry, in situ hybridization, and quantitative RT-PCR of miRNA and mRNA. miR-7 was expressed in the human developing pancreas from around 9 wga and reached its maximum expression levels between 14 and 18 wga, coinciding with the exponential increase of the pancreatic endocrine hormones. Throughout development miR-7 expression was preferentially localized to endocrine cells and its expression persisted in the adult pancreas. The present study provides a detailed analysis of the spatiotemporal expression of miR-7 in developing human pancreas. The specific localization of miR-7 expression to fetal and adult endocrine cells indicates a potential role for miR-7 in endocrine cell differentiation and/or function. Future functional studies of a potential role for miR-7 function in islet cell differentiation and physiology are likely to identify novel targets for the treatment of diabetes and will lead to the development of improved protocols for generating insulin-producing cells for cell replacement therapy.     

Isothermal sensitive detection of microRNA using an autonomous DNA machine recycling output as input

An autonomous DNA machine recycling the output as the input for isothermal, sensitive, and specific detection of miRNAs has been developed. This machine shows considerably high signal amplification efficiency (～1000-fold) and thus a low detection limit (～20 amol). The machine also shows high specificity, discriminating 50 amol of synthetic miRNA from 100-fold larger amounts of its family member and from 100 ng of unrelated total RNAs. Moreover, it is available for practically detecting natural miRNAs in total RNAs.     

Changes in root system structure,leaf water potential and gas exchange of maize and triticale seedlings affected by soil compaction

The physiological reasons associated with differential sensitivity of C₃ and C₄ plant species to soil compaction stress are not well explained and understood. The responses of growth characteristics, changes in leaf water potential and gas exchange in maize and triticale to a different soil compaction were investigated. In the present study seedlings of triticale and maize, representative of C₃ and C₄ plants were subjected to low (L – 1.10 g cm⁻³), moderate (M – 1.34 g cm⁻³) and severe (S – 1.58 g cm⁻³) soil compaction level. Distinct differences in distribution of roots in the soil profile were observed. Plants of treatments M or S in comparison to treatment L, showed a decrease in leaf number, dry mass of stem, leaves and roots, and an increase in the shoot to root ratio. A drastic decrease in root biomass in M and S treatments in the soil profile on depth from 15 to 40 cm was observed. Any level of soil compaction did not influence the number of seminal and seminal-adventitious roots but decreased their length. The number and total length of nodal roots decreased with compaction. Changes of growth traits in M and S treatments in comparison to the L were greater for maize than for triticale and were accompanied by daily changes in water potential (ψ) and gas exchange parameters (P_N, E, g_s). Differences between M and S treatments in daily changes in ψ for maize were in most cases statistically insignificant, whereas for triticale, they were statistically significant. Differences in the responses of maize and triticale to soil compaction were found in P_N, E and g_s in particular for the measurements taken at 12:00 and 16:00. The highest correlation coefficients were obtained for the relationship between leaf water potential and stomatal conductance, both for maize and triticale, which indicates the close association between stomata behavior and changes in leaf water status.     

Cloning and characterization of miRNAs from maize seedling roots under low phosphorus stress

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition in plants and animals. In this study, a small RNA library was constructed to identify conserved miRNAs as well as novel miRNAs in maize seedling roots under low level phosphorus stress. Twelve miRNAs were identified by high throughput sequencing of the library and subsequent analysis, two belong to conserved miRNA families (miRNA399b and miRNA156), and the remaining ten are novel and one of latter is conserved in gramineous species. Based on sequence homology, we predicted 125 potential target genes of these miRNAs and then expression patterns of 7 miRNAs were validated by semi-RT-PCR analysis. MiRNA399b, Zma-miR3, and their target genes (Zmpt1 and Zmpt2) were analyzed by real-time PCR. It is shown that both miRNA399b and Zma-miR3 are induced by low phosphorus stress and regulated by their target genes (Zmpt1 and Zmpt2). Moreover, Zma-miR3, regulated by two maize inorganic phosphate transporters as a newly identified miRNAs, would likely be directly involved in phosphate homeostasis, so was miRNA399b in Arabidopsis and rice. These results indicate that both conserved and maize-specific miRNAs play important roles in stress responses and other physiological processes correlated with phosphate starvation, regulated by their target genes. Identification of these differentially expressed miRNAs will facilitate us to uncover the molecular mechanisms underlying the progression of maize seedling roots development under low level phosphorus stress.     

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.