Nowadays, characterization of the vital roles of miRNAs play in plant stress responses is an active research field. Although many studies have demonstrated that plant miRNAs function as important regulators in development and morphogenesis processes, more reports are indicating that plant miRNAs are also involved in environmental stress tolerance .
Since abiotic stress is one of the primary causes of crop losses worldwide, unraveling the complex mechanisms underlying stress resistance of plants has profound significance. Recently, the newly developed sequencing technologies, such as the Illumina Genome Analyzer (GA), Roche/454 FLX system, and the ABI SOLiD system, show advances over traditional methods with improved throughput and dramatically reduced cost. Currently, applications of high-throughput sequencing technologies are arousing much research interest, such as identification of entire sets of miRNAs, which deliver new insights into the role of miRNAs in plant development, and stress related regulation. By using this method, a number of soybean miRNAs have been well annotated . Differing from microarray, high throughput sequencing allows us to comprehensively survey stress related miRNAs. To date, little is known about the functions of miRNAs in abiotic stress responses in Glycine max.
In this study, we sequenced and analyzed small RNAs of the soybean under three treatments based on deep sequencing. Investigation of the small RNAs showed that gma-miR1507a (936 627 sequence tags) was represented in our sequencing libraries. One hundred and thirty three known miRNAs and 50 novel miRNAs were obtained from next generation sequencing data. Through expression abundance of the miRNA repertoires under drought, salinity, and alkalinity stress conditions, many miRNAs were found to have a wide range of expression levels between libraries. This characteristic of variability in miRNA expression may be due to miRNA mature processing , and/or stress associated regulation [2, 36]. We envision that these miRNAs might have functional significance, suggesting they may participate in the plant stress response. Highly abundant miRNAs seem to exhibit similar conservation. For example, miR2188 and miR3522b exhibit high expression levels in all four libraries and are conserved across many species. Such observations support previous results that the most abundant miRNAs were phylogenetically conservative .
Both miRNAs and star miRNAs are generated from step-loop hairpin structures. MiRNAs are stable and participate in translational repression or cleavage of mRNA by binding or anchoring to the coding region of mRNA sequences . Khvorova et al.  inferred from the considerably low abundance of star miRNAs that these strands are typically destroyed when released from pre-miRNA stem. The low expression levels of star miRNA sequences, such as miR156d*, miR157b*, and miR3630*, further support the miRNA synthesis hypothesis. Next generation sequencing is a powerful tool in the detection of miRNA and star miRNA [[15, 39] and ]. The correlation between star miRNA and its flexible expression may reveal its particular regulated function. MiR162* and miR482* may be involved in regulating stress. Two arms of a single hairpin, giving rise to RNA function isolation by different sequences, may associate with distinct biological activities. Small novel miRNAs annotated in our study, such as the 5' and 3' of Gma-004 and Gma006, were derived from predicted hairpin structures.
Plant miRNAs have been reported as having a strong propensity towards regulating responses to abiotic stress, including dehydration, freezing, salinity, alkalinity, and other stresses by transcriptional factors or proteins . Expression levels of miRNAs induced by environmental stressors vary. They therefore may play a key role in targeting stress-regulated genes. It has been reported that stress response miRNAs were ubiquitously present in Populus , soybean , and other plants. Previous studies have reported that members of miR167, miR319, and miR393 were similarly regulated in stress tolerance [9, 42, 43]. In this study, members of miR1520n, miR4374b, and miR4396 were up-regulated simultaneously under three stresses, which implies that they might target genes that function as negative regulators of stress tolerance. In addition, it was previously reported that miR395 was previously reported to be up-regulated in a salt induced soybean line targeting sulfurylase and ASP1 genes under sulfate starvation conditions. Therefore, we speculate that miR395 might be involved in non-specific salt-induced responding pathways, such as the maintenance of energy supply [7, 13]. Moreover, miR166 is responsive to dehydration stress in barley , and it is abundant and up-regulated in soybean seedlings under dehydration conditions. MiR393a, targeting F-box proteins and a basic-helix-loop-helix family protein, was up-regulated in cold, dehydration, salt, or ABA stress , and down-regulated in soybean under alkaline stress. These responsive miRNAs are involved in post-transcriptional regulation during stress responsive processes.
Deep sequencing of the small RNA transcriptome yields an incredible amount of data, from which we can not only determine known miRNAs, but also successfully explore novel miRNAs with high accuracy and efficiency. First, in this study, we have identified 133 known and 50 novel miRNAs in Glycine max, which illustrates the diversity of miRNA expression in Glycine max, revealing the presence of more miRNAs than previously known. In addition, deep sequencing technologies in combination with bioinformatics analysis enabled us to profile the miRNA expression patterns for further miRNA functional insights, and to elucidate the underlying molecular mechanisms and diverse physiological pathways. Second, comparing miRNA expression profiles under various induced conditions, we found significant differences in miRNA regulation patterns, with 71, 50, and 45 altered expression patterns under drought, salinity, and alkalinity, respectively. The differentially expressed miRNAs obtained in this study can serve as a basis for further identification of the regulation roles of stress tolerance in Glycine max.