MicroRNA172b-5p/trehalose-6-phosphate synthase module stimulates trehalose synthesis and microRNA172b-3p/AP2-like module accelerates flowering in barley upon drought stress.

MicroRNAs (miRNAs) are major regulators of gene expression during plant development under normal and stress conditions. In this study, we analyzed the expression of 150 conserved miRNAs during drought stress applied to barley ready to flower. The dynamics of miRNAs expression was also observed after rewatering. Target messenger RNA (mRNAs) were experimentally identified for all but two analyzed miRNAs, and 41 of the targets were not reported before. Drought stress applied to barley induced accelerated flowering coordinated by a pair of two differently expressed miRNAs originating from a single precursor: hvu-miR172b-3p and hvu-miR172b-5p. Increased expression of miRNA172b-3p during drought leads to the downregulation of four APETALA2(AP2)-like genes by their mRNA cleavage. In parallel, the downregulation of the miRNA172b-5p level results in an increased level of a newly identified target, trehalose-6-phosphate synthase, a key enzyme in the trehalose biosynthesis pathway. Therefore, drought-treated plants have higher trehalose content, a known osmoprotectant, whose level is rapidly dropping after watering. In addition, trehalose-6-phosphate, an intermediate of the trehalose synthesis pathway, is known to induce flowering. The hvu-miRNA172b-5p/trehalose-6-phosphate synthase and hvu-miRNA172b-3p/AP2-like create a module leading to osmoprotection and accelerated flowering induction during drought.

Recent Insights into Plant miRNA Biogenesis: Multiple Layers of miRNA Level Regulation.

MicroRNAs are small RNAs, 20-22 nt long, the main role of which is to downregulate gene expression at the level of mRNAs. MiRNAs are fundamental regulators of plant growth and development in response to internal signals as well as in response to abiotic and biotic factors. Therefore, the deficiency or excess of individual miRNAs is detrimental to particular aspects of a plant's life. In consequence, the miRNA levels must be appropriately adjusted. To obtain proper expression of each miRNA, their biogenesis is controlled at multiple regulatory layers. Here, we addressed processes discovered to influence miRNA steady-state levels, such as MIR transcription, co-transcriptional pri-miRNA processing (including splicing, polyadenylation, microprocessor assembly and activity) and miRNA-encoded peptides synthesis. MiRNA stability, RISC formation and miRNA export out of the nucleus and out of the plant cell also define the levels of miRNAs in various plant tissues. Moreover, we show the evolutionary conservation of miRNA biogenesis core proteins across the plant kingdom.

A Functional Network of Novel Barley MicroRNAs and Their Targets in Response to Drought.

The regulation of mRNA (messenger RNA) levels by microRNA-mediated activity is especially important in plant responses to environmental stresses. In this work, we report six novel barley microRNAs, including two processed from the same precursor that are severely downregulated under drought conditions. For all analyzed microRNAs, we found target genes that were upregulated under drought conditions and that were known to be involved in a plethora of processes from disease resistance to chromatin-protein complex formation and the regulation of transcription in mitochondria. Targets for novel barley microRNAs were confirmed through degradome data analysis and RT-qPCR using primers flanking microRNA-recognition site. Our results show a broad transcriptional response of barley to water deficiency conditions through microRNA-mediated gene regulation and facilitate further research on drought tolerance in crops.

Micromanagement of Developmental and Stress-Induced Senescence: The Emerging Role of MicroRNAs.

MicroRNAs are short (19⁻24-nucleotide-long), non-coding RNA molecules. They downregulate gene expression by triggering the cleavage or translational inhibition of complementary mRNAs. Senescence is a stage of development following growth completion and is dependent on the expression of specific genes. MicroRNAs control the gene expression responsible for plant competence to answer senescence signals. Therefore, they coordinate the juvenile-to-adult phase transition of the whole plant, the growth and senescence phase of each leaf, age-related cellular structure changes during vessel formation, and remobilization of resources occurring during senescence. MicroRNAs are also engaged in the ripening and postharvest senescence of agronomically important fruits. Moreover, the hormonal regulation of senescence requires microRNA contribution. Environmental cues, such as darkness or drought, induce senescence-like processes in which microRNAs also play regulatory roles. In this review, we discuss recent findings concerning the role of microRNAs in the senescence of various plant species.

The plastid-nucleus located DNA/RNA binding protein WHIRLY1 regulates microRNA-levels during stress in barley (Hordeum vulgare L.).

In this article a novel mechanism of retrograde signaling by chloroplasts during stress is described. This mechanism involves the DNA/RNA binding protein WHIRLY1 as a regulator of microRNA levels. By virtue of its dual localization in chloroplasts and the nucleus of the same cell, WHIRLY1 was proposed as an excellent candidate coordinator of chloroplast function and nuclear gene expression. Comparison of wild-type and transgenic plants with an RNAi-mediated knockdown of WHIRLY1 showed, that the transgenic plants were unable to cope with continuous high light conditions. They were impaired in production of several microRNAs mediating post-transcriptional responses during stress. The results support a central role of WHIRLY1 in retrograde signaling and also underpin a so far underestimated role of microRNAs in this process.

Developmental changes in barley microRNA expression profiles coupled with miRNA target analysis.

MicroRNAs are 19- to 24-nt-long single-stranded RNAs that are crucial regulators of gene expression which control plant development and response to environmental cues. We have analyzed microtranscriptomes of five barley developmental stages. Generally, during the barley development, miR168-3p and miR1432-5p levels increase while the 5'U-miR156-5p level decreases (with exception for the 2-week-old barley). We have identified two miR156-5p izomiRs (called 5'U-miR156-5p [20 nt] and 5'UU-miR156-5p [21 nt]), which were expressed differently during barley development. The 5' U-miR156-5p level decreased in 3-week-, 6-week-, and 68-day-old barley, when compared to the 1-week-old plants. Meanwhile, the 5' UU-miR156-5p level increased significantly in the 68-day-old barley plants. Moreover, only the 5' U-miR156 isomiR recognizes and guides unique transcription factor mRNAs from the Squamosa Promoter Binding Protein-Like (SPL) family. We identified many non-canonical microRNAs with changed expression levels during the barley development. Here, we present the profiles of microRNA expression characteristics for particular barley developmental stages. These analyses are accompanied by the experimental degradome analysis of miRNA targets.

Barley primary microRNA expression pattern is affected by soil water availability.

MicroRNAs are short molecules of 21-24 nt in length. They are present in all eukaryotic organisms and regulate gene expression by guiding posttranscriptional silencing of mRNAs. In plants, they are key players in signal transduction, growth and development, and in response to abiotic and biotic stresses. Barley (Hordeum vulgare) is an economically important monocotyledonous crop plant. Drought is the world's main cause of loss in cereal production. We have constructed a high-throughput Real-Time RT-qPCR platform for parallel determination of 159 barley primary microRNAs' levels. The platform was tested for two drought-and-rehydration-treated barley genotypes (Rolap and Sebastian). We have determined changes in the expression of primary microRNAs responding to mild drought, severe drought, and rehydration. Based on the results obtained, we conclude that alteration in the primary microRNA expression is relative to the stress's intensity. Mild drought and rehydration mostly decrease the pri-miRNA levels in both of the tested genotypes. Severe drought mainly induces the primary microRNA expression. The main difference between the genotypes tested was a much-stronger induction of pri-miRNAs in Rolap encountering severe drought. The primary microRNAs respond dynamically to mild drought, severe drought, and rehydration treatments. We propose that some of the individual pri-miRNAs could be used as drought stress or rehydration markers. The usage of the platform in biotechnology is also postulated.

Heat Stress Affects Pi-related Genes Expression and Inorganic Phosphate Deposition/Accumulation in Barley.

Phosphorus (P) in plants is taken from soil as an inorganic phosphate (Pi) and is one of the most important macroelements in growth and development. Plants actively react to Pi starvation by the induced expression of Pi transporters, MIR399, MIR827, and miR399 molecular sponge - IPS1 genes and by the decreased expression of the ubiquitin-conjugating enzyme E2 (PHOSPHATE2 - PHO2) and Pi sensing and transport SPX-MFS genes. The PHO2 protein is involved in the degradation of Pi transporters PHT1;1 (from soil to roots) and PHO1 (from roots to shoots). The decreased expression of PHO2 leads to Pi accumulation in shoots. In contrast, the pho1 mutant shows a decreased level of Pi concentration in shoots. Finally, Pi starvation leads to decreased Pi concentration in all plant tissues. Little is known about plant Pi homeostasis in other abiotic stress conditions. We found that, during the first hour of heat stress, Pi accumulated in barley shoots but not in the roots, and transcriptomic data analysis as well as RT-qPCR led us to propose an explanation for this phenomenon. Pi transport inhibition from soil to roots is balanced by lower Pi efflux from roots to shoots directed by the PHO1 transporter. In shoots, the PHO2 mRNA level is decreased, leading to an increased Pi level. We concluded that Pi homeostasis in barley during heat stress is maintained by dynamic changes in Pi-related genes expression.

mirEX 2.0 - an integrated environment for expression profiling of plant microRNAs.

BACKGROUND: MicroRNAs are the key post-transcriptional regulators of gene expression in development and stress responses. Thus, precisely quantifying the level of each particular microRNA is of utmost importance when studying the biology of any organism. DESCRIPTION: The mirEX 2.0 web portal ( http://www.combio.pl/mirex ) provides a comprehensive platform for the exploration of microRNA expression data based on quantitative Real Time PCR and NGS sequencing experiments, covering various developmental stages, from wild-type to mutant plants. The portal includes mature and pri-miRNA expression levels detected in three plant species (Arabidopsis thaliana, Hordeum vulgare and Pellia endiviifolia), and in A. thaliana miRNA biogenesis pathway mutants. In total, the database contains information about the expression of 461 miRNAs representing 268 families. The data can be explored through the use of advanced web tools, including (i) a graphical query builder system allowing a combination of any given species, developmental stages and tissues, (ii) a modular presentation of the results in the form of thematic windows, and (iii) a number of user-friendly utilities such as a community-building discussion system and extensive tutorial documentation (e.g., tooltips, exemplary videos and presentations). All data contained within the mirEX 2.0 database can be downloaded for use in further applications in a context-based way from the result windows or from a dedicated web page. CONCLUSIONS: The mirEX 2.0 portal provides the plant research community with easily accessible data and powerful tools for application in multi-conditioned analyses of miRNA expression from important plant species in different biological and developmental backgrounds.

Transcriptionally and post-transcriptionally regulated microRNAs in heat stress response in barley.

Heat stress is one of the major abiotic factors that can induce severe plant damage, leading to a decrease in crop plant productivity. Despite barley being a cereal of great economic importance, few data are available concerning its thermotolerance mechanisms. In this work microRNAs (miRNAs) involved in heat stress response in barley were investigated. The level of selected barley mature miRNAs was examined by hybridization. Quantitative real-time PCR (RT-qPCR) was used to monitor the changes in the expression profiles of primary miRNA (pri-miRNA) precursors, as well as novel and conserved target genes during heat stress. The miRNA-mediated cleavage sites in the target transcripts were confirmed by degradome analysis and the 5' RACE (rapid amplification of cDNA ends) approach. Four barley miRNAs (miR160a, 166a, 167h, and 5175a) were found which are heat stress up-regulated at the level of both mature miRNAs and precursor pri-miRNAs. Moreover, the splicing of introns hosting miR160a and miR5175a is also heat induced. The results demonstrate transcriptional and post-transcriptional regulation of heat-responsive miRNAs in barley. The observed induction of miRNA expression is correlated with the down-regulation of the expression level of their experimentally identified new and conservative target genes.

Developmentally regulated expression and complex processing of barley pri-microRNAs.

BACKGROUND: MicroRNAs (miRNAs) regulate gene expression via mRNA cleavage or translation inhibition. In spite of barley being a cereal of great economic importance, very little data is available concerning its miRNA biogenesis. There are 69 barley miRNA and 67 pre-miRNA sequences available in the miRBase (release 19). However, no barley pri-miRNA and MIR gene structures have been shown experimentally. In the present paper, we examine the biogenesis of selected barley miRNAs and the developmental regulation of their pri-miRNA processing to learn more about miRNA maturation in barely. RESULTS: To investigate the organization of barley microRNA genes, nine microRNAs - 156g, 159b, 166n, 168a-5p/168a-3p, 171e, 397b-3p, 1120, and 1126 - were selected. Two of the studied miRNAs originate from one MIR168a-5p/168a-3p gene. The presence of all miRNAs was confirmed using a Northern blot approach. The miRNAs are encoded by genes with diverse organizations, representing mostly independent transcription units with or without introns. The intron-containing miRNA transcripts undergo complex splicing events to generate various spliced isoforms. We identified miRNAs that were encoded within introns of the noncoding genes MIR156g and MIR1126. Interestingly, the intron that encodes miR156g is spliced less efficiently than the intron encoding miR1126 from their specific precursors. miR397b-3p was detected in barley as a most probable functional miRNA, in contrast to rice where it has been identified as a complementary partner miRNA*. In the case of miR168a-5p/168a-3p, we found the generation of stable, mature molecules from both pre-miRNA arms, confirming evolutionary conservation of the stability of both species, as shown in rice and maize. We suggest that miR1120, located within the 3' UTR of a protein-coding gene and described as a functional miRNA in wheat, may represent a siRNA generated from a mariner-like transposable element. CONCLUSIONS: Seven of the eight barley miRNA genes characterized in this study contain introns with their respective transcripts undergoing developmentally specific processing events prior to the dicing out of pre-miRNA species from their pri-miRNA precursors. The observed tendency to maintain the intron encoding miR156g within the transcript, and preferences in splicing the miR1126-harboring intron, may suggest the existence of specific regulation of the levels of intron-derived miRNAs in barley.

Uncoupling protein 1 inhibition by purine nucleotides is under the control of the endogenous ubiquinone redox state.

We studied non-esterified fatty acid-induced uncoupling of heterologously expressed rat UCP1 (uncoupling protein 1) in yeast mitochondria, as well as UCP1 in rat BAT (brown adipose tissue) mitochondria. The proton-conductance curves and the relationship between the ubiquinone reduction level and membrane potential were determined in non-phosphorylating BAT and yeast mitochondria. The ADP/O method was applied to determine the ADP phosphorylation rate and the relationship between the ubiquinone reduction level and respiration rate in yeast mitochondria. Our studies of the membranous ubiquinone reduction level in mitochondria demonstrate that activation of UCP1 leads to a purine nucleotide-sensitive decrease in the ubiquinone redox state. Results obtained for non-phosphorylating and phosphorylating mitochondria, as the endogenous ubiquinone redox state was gradually varied by a lowering rate of the ubiquinone-reducing or ubiquinol-oxidizing pathways, indicate that the endogenous ubiquinone redox state has no effect on non-esterified fatty acid-induced UCP1 activity in the absence of GTP, and can only regulate this activity through sensitivity to inhibition by the purine nucleotide. At a given oleic acid concentration, inhibition by GTP diminishes when ubiquinone is reduced sufficiently. The ubiquinone redox state-dependent alleviation of UCP1 inhibition by the purine nucleotide was observed at a high ubiquinone reduction level, when it exceeded 85-88%.