microRNA profiling of root tissues and root forming explant cultures in Medicago truncatula

Plant root architecture is regulated by the initiation and modulation of cell division in regions containing pluripotent stem cells known as meristems. In roots, meristems are formed early in embryogenesis, in the case of the root apical meristem (RAM), and during organogenesis at the site of lateral root or, in legumes, nodule formation. Root meristems can also be generated in vitro from leaf explants cultures supplemented with auxin. microRNAs (miRNAs) have emerged as regulators of many key biological functions in plants including root development. To identify key miRNAs involved in root meristem formation in Medicago truncatula, we used deep sequencing to compare miRNA populations. Comparisons were made between: (1) the root tip (RT), containing the RAM and the elongation zone (EZ) tissue and (2) root forming callus (RFC) and non-root forming callus (NRFC). We identified 83 previously reported miRNAs, 24 new to M. truncatula, in 44 families. For the first time in M. truncatula, members of conserved miRNA families miR165, miR181 and miR397 were found. Bioinformatic analysis identified 38 potential novel miRNAs. Selected miRNAs and targets were validated using Taqman miRNA assays and 5′ RACE. Many miRNAs were differentially expressed between tissues, particularly RFC and NRFC. Target prediction revealed a number of miRNAs to target genes previously shown to be differentially expressed between RT and EZ or RFC and NRFC and important in root development. Additionally, we predict the miRNA/target relationships for miR397 and miR160 to be conserved in M. truncatula. Amongst the predictions, were AUXIN RESPONSE FACTOR 10, targeted by miR160 and a LACCASE-like gene, targeted by miR397, both are miRNA/target pairings conserved in other species.     

Identification of Nitrogen Starvation-Responsive MicroRNAs in Arabidopsis thaliana

microRNAs (miRNAs) are a class of negative regulators that take part in many processes such as growth and development, stress responses, and metabolism in plants. Recently, miRNAs were shown to function in plant nutrient metabolism. Moreover, several miRNAs were identified in the response to nitrogen (N) deficiency. To investigate the functions of other miRNAs in N deficiency, deep sequencing technology was used to detect the expression of small RNAs under N-sufficient and -deficient conditions. The results showed that members from the same miRNA families displayed differential expression in response to N deficiency. Upon N starvation, the expression of miR169, miR171, miR395, miR397, miR398, miR399, miR408, miR827, and miR857 was repressed, whereas those of miR160, miR780, miR826, miR842, and miR846 were induced. miR826, a newly identified N-starvation-induced miRNA, was found to target the AOP2 gene. Among these N-starvation-responsive miRNAs, several were involved in cross-talk among responses to different nutrient (N, P, S, Cu) deficiencies. miR160, miR167, and miR171 could be responsible for the development of Arabidopsis root systems under N-starvation conditions. In addition, twenty novel miRNAs were identified and nine of them were significantly responsive to N-starvation. This study represents comprehensive expression profiling of N-starvation-responsive miRNAs and advances our understanding of the regulation of N homeostasis mediated by miRNAs.     

In silico Characterisation and Phylogenetic Analysis of Two Evolutionarily Conserved miRNAs (miR166 and miR171) from Black Pepper (Piper nigrum L.)

Among computationally predicted and experimentally validated plant miRNAs, several are conserved across species boundaries in the plant kingdom. In this study, a combined experimental–in silico approach was adopted for characterization of two conserved miRNAs, miR166 and miR171, from black pepper (Piper nigrum). A PCR-based detection and cloning strategy of miRNAs from tissues of black pepper was used. Conservation analysis of miR166 and miR171 along with their corresponding targets identified from P. nigrum revealed that these miRNAs are highly conserved with their counterparts in other plant species. miRNA-mediated cleavage of the conserved targets was also verified by RLM-RACE experiments. Real-time quantitative PCR revealed the differential expression patterns of these miRNAs in black pepper tissues. Our miRNA-based phylogenetic analysis of plants belonging to the Piperaceae family was in agreement with the typical paleoherb evolutionary scheme of primitive angiosperms. This method will help in the detection of evolutionarily conserved miRNAs in other plant species and provide a strategy for a novel phylogenetic reconstruction based on the evolutionary history of miRNA genes.     

Regulation of miR319 during cold stress in sugarcane

MicroRNAs (miRNAs) are part of a novel mechanism of gene regulation that is active in plants under abiotic stress conditions. In the present study, 12 miRNAs were analysed to identify miRNAs differentially expressed in sugarcane subjected to cold stress (4 °C). The expression of miRNAs assayed by stem–loop RT‐PCR showed that miR319 is up‐regulated in sugarcane plantlets exposed to 4 °C for 24 h. The induction of miR319 expression during cold stress was observed in both roots and shoots. Sugarcane miR319 was also regulated by treatment with abscisic acid. Putative targets of this miRNA were identified and their expression levels were decreased in sugarcane plantlets exposed to cold. The cleavage sites of two targets were mapped using a 5′ RACE PCR assay confirming the regulation of these genes by miR319. When sugarcane cultivars contrasting in cold tolerance were subjected to 4 °C, we observed up‐regulation of miR319 and down‐regulation of the targets in both varieties; however, the changes in expression were delayed in the cold‐tolerant cultivar. These results suggest that differences in timing and levels of the expression of miR319 and its targets could be tested as markers for selection of cold‐tolerant sugarcane cultivars.     

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.     

The role of the miR399-<Emphasis Type="Italic">PHO2</Emphasis> module in the regulation of flowering time in response to different ambient temperatures in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A moderate change in ambient temperature significantly affects plant physiology including flowering time. MiR399 and its target gene PHOSPHATE 2 (PHO2) are known to play a role in the maintenance of phosphate homeostasis. However, the regulation of flowering time by the miR399-PHO2 module has not been investigated. As we have previously identified miR399 as an ambient temperature-responsive miRNA, we further investigated whether a change in expression of the miR399-PHO2 module affects flowering time in response to ambient temperature changes. Here, we showed that miR399b-overexpressing plants and a loss-of-function allele of PHO2 (pho2) exhibited an early flowering phenotype only at normal temperature (23°C). Interestingly, their flowering time at lower temperature (16°C) was similar to that of wild-type plants, suggesting that alteration in flowering time by miR399 and its target PHO2 was seen only at normal temperature (23°C). Flowering time ratio (16°C/23°C) revealed that miR399b-overexpressing plants and pho2 mutants showed increased sensitivity to ambient temperature changes. Expression analysis indicated that expression of TWIN SISTER OF FT (TSF) was increased in miR399b-overexpressing plants and pho2 mutants at 23°C, suggesting that their early flowering phenotype is associated with TSF upregulation. Taken together, our results suggest that miR399, an ambient temperature-responsive miRNA, plays a role in ambient temperature-responsive flowering in Arabidopsis.     

miRNA profiling in leaf and cork tissues of Quercus suber reveals novel miRNAs and tissue-specific expression patterns

The differentiation of cork (phellem) cells from the phellogen (cork cambium) is a secondary growth process observed in the cork oak tree conferring a unique ability to produce a thick layer of cork. At present, the molecular regulators of phellem differentiation are unknown. The previously documented involvement of microRNAs (miRNAs) in the regulation of developmental processes, including secondary growth, motivated the search for these regulators in cork oak tissues. We performed deep sequencing of the small RNA fraction obtained from cork oak leaves and differentiating phellem. RNA sequences with lengths of 19–25 nt derived from the two libraries were analysed, leading to the identification of 41 families of conserved miRNAs, of which the most abundant were miR167, miR165/166, miR396 and miR159. Thirty novel miRNA candidates were also unveiled, 11 of which were unique to leaves and 13 to phellem. Northern blot detection of a set of conserved and novel miRNAs confirmed their differential expression profile. Prediction and analysis of putative miRNA target genes provided clues regarding processes taking place in leaf and phellem tissues, but further experimental work will be needed for functional characterization. In conclusion, we here provide a first characterization of the miRNA population in a Fagacea species, and the comparative analysis of miRNA expression in leaf and phellem libraries represents an important step to uncovering specific regulatory networks controlling phellem differentiation.     

MicroRNAs in the shoot apical meristem of soybean

Plant microRNAs (miRNAs) play crucial regulatory roles in various developmental processes. In this study, we characterize the miRNA profile of the shoot apical meristem (SAM) of an important legume crop, soybean, by integrating high-throughput sequencing data with miRNA microarray analysis. A total of 8423 non-redundant sRNAs were obtained from two libraries derived from micro-dissected SAM or mature leaf tissue. Sequence analysis allowed the identification of 32 conserved miRNA families as well as 8 putative novel miRNAs. Subsequent miRNA profiling with microarrays verified the expression of the majority of these conserved and novel miRNAs. It is noteworthy that several miRNAs* were expressed at a level similar to or higher than their corresponding mature miRNAs in SAM or mature leaf, suggesting a possible biological function for the star species. In situ hybridization analysis revealed a distinct spatial localization pattern for a conserved miRNA, miR166, and its star speciessuggesting that they serve different roles in regulating leaf development. Furthermore, localization studies showed that a novel soybean miRNA, miR4422a, was nuclear-localized. This study also indicated a novel expression pattern of miR390 in soybean. Our approach identified potential key regulators and provided vital spatial information towards understanding the regulatory circuits in the SAM of soybean during shoot development.     

盐胁迫下大豆根组织定量PCR分析中内参基因的选择

实时荧光定量PCR已广泛用于基因表达的分析, 适当的内参基因选择是获得准确分析结果的关键。在大豆(Glycine max)分子生物学研究中, 逆境响应基因和microRNA (miRNA)表达的内参辅助检测基因均有哪些目前尚不清楚。该研究选用不同盐梯度和时间点组合处理的大豆根组织为材料, 对已报道的其它条件下表达相对稳定的内参基因(ACT、ACT2/7、CYP2、ELF1A、ELF1B、F-Box、TUA和UBC2)以及miRNA内参基因(U6、miR1515a、miR1520c、miR1520d、miR171a和miR171b)的表达情况进行了全面检测; 并采用Δ-Ct、Bestkeeper、NormFinder和Genorm四种方法对检测结果进行了综合分析, 发现ELF1B和CYP2适合作为大豆根系盐胁迫响应基因研究的内参基因, miR1515a和U6适合作为盐胁迫下大豆根组织miRNA研究的内参。上述研究结果为大豆盐胁迫响应基因和miRNA表达及其进一步的功能研究奠定了基础。