MicroRNAs (miRNAs) are a class of non-coding small RNAs (smRNAs) that act to reduce expression of target genes by interacting with their target mRNAs in a sequence-specific manner. Since their discovery it has become clear that miRNAs are an important component in the regulation of many genes in most eukaryotic cells. In plants, most currently validated miRNA targets code for transcription factor families with crucial developmental functions, including the control of root and shoot architecture, vegetative to reproductive phase transitions and leaf and flower morphogenesis [1
miRNAs are processed from a primary miRNA transcript which folds to form an imperfect stem-loop. The pri-miRNA hairpin is recognised and processed to a smRNA duplex consisting of the miRNA and complementary miRNA* by a protein complex containing a DCL1-type RNase. The mature miRNA, which is typically 20–21 nt in length, is then incorporated into the RNA Induced Silencing Complex (RISC) to regulate one or more target genes in trans
through a base pairing mechanism. Most plant miRNAs appear to trigger both mRNA cleavage (between the nucleotides matching the 10th
position of the miRNA) and translational repression of their target genes [3
]. Although these two mechanisms are additive, they can be dissociated when slicing activity is disabled by a mis-pairing in the central region between the miRNA and its target [4
]. In plants, the high level of complementarity between the miRNAs and their targets suggests slicing is the predominant mode of action of miRNAs [7
]. Alternatively, miRNAs can regulate their target indirectly through the production of trans-acting short interfering RNAs (tasiRNAs) [8
]. tasiRNAs are synthesised from a non-coding mRNA that is processed to phased 21 nt smRNAs by a miRNA triggered process. Like miRNAs, tasiRNAs can regulate multiple target genes through a slicing mechanism.
The number of annotated miRNAs in miRBase has exponentially increased in the last decade [10
The earliest group of miRNAs were identified in silico
using algorithms to predict stem-loop precursors and targets present in the genome and/or EST databases [11
]. Subsequent developments in high throughput sequencing made it possible to identify miRNAs based on sequencing of smRNA libraries in a wide range of species. Schreiber et al.
] identified 100 miRNAs, including 44 new miRNAs, from barley leaves using short-read sequence data. A major challenge of sequencing based approaches is to identify the miRNAs amongst a smRNA population mostly composed of short-interfering RNAs (siRNAs) [17
]. Distinguishing these two major smRNA classes relies principally on identifying their origin. An siRNA locus produces several overlapping siRNAs, whereas the pri-miRNA encoded by a MIR
gene usually produces one miRNA from an imperfect RNA hairpin [18
]. Additional criteria can also help classify a smRNA, such as its length and mode of action. Most miRNAs and tasiRNAs are 21 nt in length and post-transcriptionally regulate their target genes in trans
, whereas the vast majority of the 24 nt smRNAs correspond to cis-acting siRNAs (casiRNAs) that regulate the transcription of their own locus of origin through a DNA methylation based mechanism.
miRNA targets are often validated using a modified 5’RACE technique to detect the products of miRNA-mediated cleavage [19
]. For most currently annotated miRNA targets, cleavage has not been verified and therefore the function of the corresponding miRNA in vivo
has not been established. Recently, techniques which combine 5’RACE and high throughput sequencing (Parallel Analysis of RNA Ends (PARE) and equivalent methods [20
]), have been used to simultaneously validate all sliced miRNA targets in a given RNA extract. Such an approach has been successfully carried out in Arabidopsis, rice, soybean, grapevine, citrus and medicago [23
]. However, identifying a miRNA regulation is dependent on examining the appropriate tissue and developmental stage. As miRNAs are predominantly post-transcriptional regulators [29
], the impact of their regulation depends on the overlap of their spatio-temporal expression with that of their target genes [1
]. miRNAs from the same family can potentially have different functions depending on their expression profile, as suggested for members of the miR169 and miR171 families that differentially accumulate in response to abiotic stress in rice [31
Despite the growing knowledge of miRNA functions in plants, only the functions of highly conserved miRNAs have been investigated in crop species. Perhaps the best characterized miRNAs in cereals are miR156 and miR172 which regulate SPL
(Squamosa Promoter-binding protein-Like
) and AP2
-like genes, respectively. miR156 controls shoot branching in rice and maize [33
] and miR172 regulates floral organ identity in rice, maize and barley [36
]. In maize, miR172 accumulation is affected by miR156 and both miRNAs are involved in the regulation of the juvenile to adult phase transition [33
]. In contrast to the highly conserved miRNAs, the majority of the newly discovered miRNAs are weakly expressed and only found in closely related species, suggesting that they have recently evolved and could contribute to determining species-specific traits.
Barley is the fourth most cultivated crop worldwide; its grains are used for both human consumption and livestock feed. From anthesis, it takes approximately 40
days to form a mature grain composed of 3 principal tissues: the embryo, the endosperm (starchy endosperm and aleurone layers), and the outside layer (seed coat and pericarp). The development of the grain can be divided in three principal stages based on morphological changes, metabolite accumulation and transcriptome analysis: pre-storage, storage (or maturation) and desiccation [42
]. The pre-storage phase, which corresponds to the first 5 Days Post Anthesis (DPA), is characterized by extensive mitotic activity in both embryo and endosperm. The transition to the storage phase, roughly between 5 and 10 DPA, can be considered as an intermediate stage characterized by dramatic transcriptional changes in order to mobilize energy resources and initiate the differentiation of the tissues that will constitute the mature grain. Throughout the maturation phase, which lasts up to ~25 DPA, aleurone and embryonic tissues acquire desiccation tolerance whereas the endosperm cells undergo endoreduplication and accumulate storage metabolites (mainly starch and proteins) [46
In this study we investigated the miRNA-mediated gene regulation that takes place during the growth of the barley grain. Since the early stages of development play a key role in determining grain quality characteristics, we focused on the pre-storage and early storage phases (0–15 DPA). From analysis of smRNA and degradome libraries, 96 genes regulated by miRNA-mediated cleavage were identified including transcription factors, kinases, oxidoreductases, hydrolases, transferases, receptors and transporters. Our data suggest that miRNAs contribute widely to the control of development of the cereal grain, notably through the regulation of phytohormone response pathways.