MicroRNAs (miRNAs) are a class of small non-protein-coding RNAs generated from single-stranded precursors with unique hairpin structures. They regulate the expression of mRNAs by targeting transcripts for cleavage or translational repression [1
]. The miRNAs were initially isolated in Caenorhabditis elegans
as developmental timing regulators [2
]. Since then, they have been found in a broad range of plants, as well as viruses and mammals. Plants miRNAs were first identified in Arabidopsis
by different research groups [3
]. The biogenesis of plant miRNAs is a complex multi-step enzymatic process [6
]. The miRNAs are initially transcribed by RNA polymerase II in the cell nucleus as long primary miRNAs that are cleaved into the miRNA:miRNA* duplexes by Dicer-like 1 (DCL1). Export of the duplexes into the cell cytoplasm is mediated by HASTY. After methyl groups are added to the 3' ends of the duplexes catalyzed by HEN1, one strand of the duplexes is selectively incorporated into the RNA-induced silencing complex (RISC) to form the mature miRNAs, whereas the other strand, designated the miRNA*, is typically degraded. DCL4 has also been shown to play a role in the biogenesis of a few miRNAs with long hairpin precursors [9
]. Plant miRNAs exhibit high complementarity to their targets and can direct RISC-mediated cleavage of target mRNAs. Thus, it is widely accepted that plant miRNAs induce post-transcriptional gene silencing predominantly through guiding mRNA cleavage. Recent studies, however, have showed that translational inhibition is another kind of action mechanism for miRNAs in plants [10
Although initial studies have largely demonstrated the role of plant miRNAs in development and morphogenesis processes, there are increasing number of reports indicating that plant miRNAs also target genes involved in biotic as well as abiotic stress responses [1
]. Low temperature is one of the most important environmental stimuli that affect plant growth and development. Recently, more and more reports demonstrate the role of plant miRNAs in cold stress response. The first study in this area, performed by Sunkar and Zhu, revealed the induced expression of miR393, miR397b and miR402 in response to cold stress as well as other kinds of stress treatment. In this study, miR319c appeared to be up-regulated by cold but not by dehydration, NaCl, or ABA treatment [16
]. Zhou et al
. (2008) identified cold up-regulated miRNAs using a computational, transcriptome-based approach [17
]. Liu et al
. (2008) and Lu et al
. (2008) used microarray analysis to identify cold-responsive miRNAs in Arabidopsis
, respectively [18
]. These studies indicate that the expression of several miRNAs is affected by the cold treatment. Despite these efforts, our knowledge of the role played by miRNAs in plant cold stress response is still limited at the whole-genome level.
The most challenging problem in understanding plant miRNAs is to identify more novel miRNAs. Three major approaches have been used for miRNA discovery in plants: forward genetics, bioinformatic prediction as well as direct cloning and sequencing. Only a few miRNAs were identified by forward genetic studies [10
] and predicting species-specific miRNAs using bioinformatics method is difficult. Thus, direct cloning and sequencing is the most effective method for plant miRNA discovery. Only a few hundred miRNAs have been identified with this approach, which leads to a premature conclusion that the types of miRNAs in plants are limited. Recently, the deep sequencing approach appeared and allowed the identification of numerous small RNAs [9
]. It not only revealed a lot more species-specific miRNAs, but also provided a picture of the genomic landscape of small RNAs.
Although significant progress has been made in identifying plant miRNAs and understanding their action mechanism, the discovery of novel miRNAs in plants on a genome-wide scale is still at the preliminary stage. One limiting factor in miRNA discovery is the availability of the whole genome sequence, only with which a comprehensive analysis of the potential hairpin precursor structures of cloned small RNAs can be performed to distinguish miRNAs from other kinds of small RNAs. Thus, most of the studies have been done in Arabidopsis
, rice and Populus
, whose whole genome sequences are known. Many evolutionarily or economically important species have not been examined yet. To further understand the function of plant miRNAs, more effort should be directed toward plant species with specific developmental features, which may contain miRNAs that are specific for these features [27
]. Several economically important winter-habit monocots, such as winter wheat, barley, oat, rye, as well as cool-season biofuel and forage grasses, can cold-acclimate and acquire high tolerance to low temperature. Although it has been shown that miRNAs are involved in plant cold stress response, little work has been done for monocotyledonous plant, especially for these winter-habit monocots, probably because rice, the model plant for monocots, is a tropical plant and incapable of cold acclimation.
has emerged as a new monocot model plant, especially for temperate cereals and related grasses [28
not only has a closer evolutionary relationship with cool-season temperate cereals and grasses than rice, but also possesses growth and developmental features that are common to these plants. As a widely distributed winter-habit temperate plant, it has vernalization requirement and is capable of cold acclimation [30
]. Recently, the draft genome sequence of Brachypodium
has been released http://www.brachypodium.org/
, which makes this plant a good model for performing whole-genome-wide study of miRNAs involved in cold response of winter-habit temperate cereals and grasses. Here we sequenced small RNA populations in Brachypodium
with and without cold stress treatment using Solexa, the high-throughput sequencing technology. Our studies not only identified species-specific miRNAs for the novel monocotyledonous model plant, but also provided useful information for cold-responsive miRNAs in temperate cereals and grasses.