Summary: Drought is a normal and recurring climate feature in most parts of the world and plays a major role in limiting crop productivity. However, plants have their own defence systems to cope with adverse climatic conditions. One of these defence mechanisms is the reprogramming of gene expression by microRNAs (miRNAs). miRNAs are small noncoding RNAs of approximately 22 nucleotides length, which have emerged as important regulators of genes at post‐transcriptional levels in a range of organisms. Some miRNAs are functionally conserved across plant species and are regulated by drought stress. These properties suggest that miRNA‐based genetic modifications have the potential to enhance drought tolerance in cereal crops. This review summarizes the current understanding of the regulatory mechanisms of plant miRNAs, involvement of plant miRNAs in drought stress responses in barley (Hordeum vulgare L.), wheat (Triticum spp.) and other plant species, and the involvement of miRNAs in plant‐adaptive mechanisms under drought stress. Potential strategies and directions for future miRNA research and the utilization of miRNAs in the improvement of cereal crops for drought tolerance are also discussed.

Summary: Drought is a major constraint to crop production, and microRNAs (miRNAs) play an important role in plant drought tolerance. Analysis of miRNAs and other classes of small RNAs (sRNAs) in barley grown under water and drought conditions reveals that drought selectively regulates expression of miRNAs and other classes of sRNAs. Low‐expressed miRNAs and all repeat‐associated siRNAs (rasiRNAs) tended towards down‐regulation, while tRNA‐derived sRNAs (tsRNAs) had the tendency to be up‐regulated, under drought. Antisense sRNAs (putative siRNAs) did not have such a tendency under drought. In drought‐tolerant transgenic barley overexpressing DREB transcription factor, most of the low‐expressed miRNAs were also down‐regulated. In contrast, tsRNAs, rasiRNAs and other classes of sRNAs were not consistently expressed between the drought‐treated and transgenic plants. The differential expression of miRNAs and siRNAs was further confirmed by Northern hybridization and quantitative real‐time PCR (qRT‐PCR). Targets of the drought‐regulated miRNAs and siRNAs were predicted, identified by degradome libraries and confirmed by qRT‐PCR. Their functions are diverse, but most are involved in transcriptional regulation. Our data provide insight into the expression profiles of miRNAs and other sRNAs, and their relationship under drought, thereby helping understand how miRNAs and sRNAs respond to drought stress in cereal crops.

Background: MicroRNAs are important components of the regulatory network of biological systems and thousands have been discovered in both animals and plants. Systematic investigations performed in species with sequenced genomes such as Arabidopsis, rice, poplar and Brachypodium have provided insights into the evolutionary relationships of this class of small RNAs among plants. However, miRNAs from barley, one of the most important cereal crops, remain unknown. Results: We performed a large scale study of barley miRNAs through deep sequencing of small RNAs extracted from leaves of two barley cultivars. By using the presence of miRNA precursor sequences in related genomes as one of a number of supporting criteria, we identified up to 100 miRNAs in barley. Of these only 56 have orthologs in wheat, rice or Brachypodium that are known to be expressed, while up to 44 appear to be specifically expressed in barley. Conclusions: Our study, the first large scale investigation of small RNAs in barley, has identified up to 100 miRNAs. We demonstrate that reliable identification of miRNAs via deep sequencing in a species whose genome has not been sequenced requires a more careful analysis of sequencing errors than is commonly performed. We devised a read filtering procedure for dealing with errors. In addition, we found that the use of a large dataset of almost 35 million reads permits the use of read abundance distributions along putative precursor sequences as a practical tool for isolating miRNAs in a large background of reads originating from other non-coding and coding RNAs. This study therefore provides a generic approach for discovering novel miRNAs where no genome sequence is available.

Background: miR399 and miR827 are both involved in conserved phosphorus (P) deficiency signalling pathways. miR399 targets the PHO2 gene encoding E2 enzyme that negatively regulates phosphate uptake and root-to-shoot allocation, while miR827 targets SPX-domain-containing genes that negatively regulate other P-responsive genes. However, the response of miR399 and miR827 to P conditions in barley has not been investigated. Results: In this study, we investigated the expression profiles of miR399 and miR827 in barley (Hordeum vulagre L.) under P-deficient and P-sufficient conditions. We identified 10 members of the miR399 family and one miR827 gene in barley, all of which were significantly up-regulated under deficient P. In addition, we found many isomirs of the miR399 family and miR827, most of which were also significantly up-regulated under deficient P. Several isomirs of miR399 members were found to be able to cleave their predicted targets in vivo. Surprisingly, a few small RNAs (sRNAs) derived from the single-stranded loops of the hairpin structures of MIR399b and MIR399e-1 were also found to be able to cleave their predicted targets in vivo. Many antisense sRNAs of miR399 and a few for miR827 were also detected, but they did not seem to be regulated by P. Intriguingly, the lowest expressed member, hvu-miR399k, had four-fold more antisense sRNAs than sense sRNAs, and furthermore under P sufficiency, the antisense sRNAs are more frequent than the sense sRNAs. We identified a potential regulatory network among miR399, its target HvPHO2 and target mimics HvIPS1 and HvIPS2 in barley under P-deficient and P-sufficient conditions. Conclusions: Our data provide an important insight into the mechanistic regulation and function of miR399, miR827 and their isomirs in barley under different P conditions.

For accurate and reliable gene expression analysis using quantitative real-time reverse transcription PCR (qPCR), the selection of appropriate reference genes as an internal control for normalization is crucial. We hypothesized that non-coding, small nucleolar RNAs (snoRNAs) would be stably expressed in different barley varieties and under different experimental treatments, in different tissues and at different developmental stages of plant growth and therefore might prove to be suitable reference genes for expression analysis of both microRNAs (miRNAs) and mRNAs. In this study, we examined the expression stability of ten candidate reference genes in six barley genotypes under five experimental stresses, drought, fungal infection, boron toxicity, nutrient deficiency and salinity. We compared four commonly used housekeeping genes; Actin (ACT), alpha-Tubulin (α-TUB), Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ADP-ribosylation factor 1-like protein (ADP), four snoRNAs; (U18, U61, snoR14 and snoR23) and two microRNAs (miR168, miR159) as candidate reference genes. We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples. For accurate and reliable gene expression analysis using quantitative real-time reverse transcription PCR (qPCR), the selection of appropriate reference genes as an internal control for normalization is crucial. We hypothesized that non-coding, small nucleolar RNAs (snoRNAs) would be stably expressed in different barley varieties and under different experimental treatments, in different tissues and at different developmental stages of plant growth and therefore might prove to be suitable reference genes for expression analysis of both microRNAs (miRNAs) and mRNAs. In this study, we examined the expression stability of ten candidate reference genes in six barley genotypes under five experimental stresses, drought, fungal infection, boron toxicity, nutrient deficiency and salinity. We compared four commonly used housekeeping genes; Actin (ACT), alpha-Tubulin (α-TUB), Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ADP-ribosylation factor 1-like protein (ADP), four snoRNAs; (U18, U61, snoR14 and snoR23) and two microRNAs (miR168, miR159) as candidate reference genes. We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples. Additionally, we found that miR168 was a suitable reference gene for expression analysis in barley. Finally, we validated the performance of our stable and unstable candidate reference genes for both mRNA and miRNA qPCR data normalization under different stress conditions and demonstrated the superiority of the stable candidates. Our data demonstrate the suitability of barley snoRNAs and miRNAs as potential reference genes for miRNA and mRNA qPCR data normalization under different stress treatments.