PEP444c encoded within the MIR444c gene regulates microRNA444c accumulation in barley.

MicroRNAs are small, noncoding RNA molecules that regulate the expression of their target genes. The MIR444 gene family is present exclusively in monocotyledons, and microRNAs444 from this family have been shown to target certain MADS-box transcription factors in rice and barley. We identified three barley MIR444 (MIR444a/b/c) genes and comprehensively characterised their structure and the processing pattern of the primary transcripts (pri-miRNAs444). Pri-microRNAs444 undergo extensive alternative splicing, generating functional and nonfunctional pri-miRNA444 isoforms. We show that barley pri-miRNAs444 contain numerous open reading frames (ORFs) whose transcripts associate with ribosomes. Using specific antibodies, we provide evidence that selected ORFs encoding PEP444a within MIR444a and PEP444c within MIR444c are expressed in barley plants. Moreover, we demonstrate that CRISPR-associated endonuclease 9 (Cas9)-mediated mutagenesis of the PEP444c-encoding sequence results in a decreased level of PEP444 transcript in barley shoots and roots and a 5-fold reduced level of mature microRNA444c in roots. Our observations suggest that PEP444c encoded by the MIR444c gene is involved in microRNA444c biogenesis in barley.

Excess nitrogen responsive HvMADS27 transcription factor controls barley root architecture by regulating abscisic acid level.

Nitrogen (N) is an important element for plant growth and development. Although several studies have examined plants' response to N deficiency, studies on plants' response to excess N, which is common in fertilizer-based agrosystems, are limited. Therefore, the aim of this study was to examine the response of barley to excess N conditions, specifically the root response. Additionally, genomic mechanism of excess N response in barley was elucidated using transcriptomic technologies. The results of the study showed that barley MADS27 transcription factor was mainly expressed in the roots and its gene contained N-responsive cis-regulatory elements in the promoter region. Additionally, there was a significant decrease in HvMADS27 expression under excess N condition; however, its expression was not significantly affected under low N condition. Phenotypic analysis of the root system of HvMADS27 knockdown and overexpressing barley plants revealed that HvMADS27 regulates barley root architecture under excess N stress. Further analysis of wild-type (WT) and transgenic barley plants (hvmads27 kd and hvmads27 c-Myc OE) revealed that HvMADS27 regulates the expression of HvBG1 ß-glucosidase, which in turn regulates abscisic acid (ABA) level in roots. Overall, the findings of this study showed that HvMADS27 expression is downregulated in barley roots under excess N stress, which induces HvBG1 expression, leading to the release of ABA from ABA-glucose conjugate, and consequent shortening of the roots.

Barley microRNAs as metabolic sensors for soil nitrogen availability.

Barley (Hordeum vulgare) is one of the most important crops in the world, ranking 4th in the worldwide production. Crop breeders are facing increasing environmental obstacles in the field, such as drought, salinity but also toxic over fertilization which not only impacts quality of the grain but also an yield. One of the most prevalent mechanisms of gene expression regulation in plants is microRNA-mediated silencing of target genes. We identified 13 barley microRNAs and 2 microRNAs* that are nitrogen excess responsive. Four microRNAs respond only in root, eight microRNAs only in shoot and one displays broad response in roots and shoots. We demonstrate that 2 microRNAs* are induced in barley shoot by nitrogen excess. For all microRNAs we identified putative target genes and confirmed microRNA-guided cleavage sites for ten out of thirteen mRNAs. None of the identified microRNAs or their target genes is known as nitrogen excess responsive. Analysis of expression pattern of thirteen target mRNAs and their cognate microRNAs showed expected correlations of their levels. The plant microRNAs analyzed are also known to respond to nitrogen deprivation and exhibit the opposite expression pattern when nitrogen excess/deficiency conditions are compared. Thus, they can be regarded as metabolic sensors of the regulation of nitrogen homeostasis in plants.

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.

miRNA Detection by Stem-Loop RT-qPCR in Studying microRNA Biogenesis and microRNA Responsiveness to Abiotic Stresses.

This chapter is devoted to a PCR-based method for analyzing the expression level of mature miRNAs which utilizes the TaqMan® technology. Stem-loop RT-qPCR requires preparation of separate cDNA templates for each analyzed miRNA as reverse transcription occurs in the presence of a miRNA-specific stem-loop reverse primer. In quantitative analysis, SYBR® Green is not used but the more sensitive TaqMan® probe that on 5' end contains a covalently attached fluorophore and on 3' quencher. When quencher and fluorophore are spatially separated due to nucleolytic DNA polymerase activity, the signal is released and quantified. This section provides a detailed and comprehensive protocol allowing for the successful analysis of mature miRNA levels in analyzed sample. Reverse transcription combined with classic real-time PCR as well as ddPCR™ (Droplet Digital™ PCR) will be presented.

Plant Science View on Biohybrid Development.

Biohybrid consists of a living organism or cell and at least one engineered component. Designing robot-plant biohybrids is a great challenge: it requires interdisciplinary reconsideration of capabilities intimate specific to the biology of plants. Envisioned advances should improve agricultural/horticultural/social practice and could open new directions in utilization of plants by humans. Proper biohybrid cooperation depends upon effective communication. During evolution, plants developed many ways to communicate with each other, with animals, and with microorganisms. The most notable examples are: the use of phytohormones, rapid long-distance signaling, gravity, and light perception. These processes can now be intentionally re-shaped to establish plant-robot communication. In this article, we focus on plants physiological and molecular processes that could be used in bio-hybrids. We show phototropism and biomechanics as promising ways of effective communication, resulting in an alteration in plant architecture, and discuss the specifics of plants anatomy, physiology and development with regards to the bio-hybrids. Moreover, we discuss ways how robots could influence plants growth and development and present aims, ideas, and realized projects of plant-robot biohybrids.

MicroRNA-mediated regulation of flower development in grasses.

Flower structure in grasses is very unique. There are no petals or sepals like in eudicots but instead flowers develop bract-like structures - palea and lemma. Reproductive organs are enclosed by round lodicule that not only protects reproductive organs but also plays an important role during flower opening. The first genetic model for floral organ development was proposed 25 years ago and it was based on the research on model eudicots. Since then, studies have been carried out to answer the question whether this model could be applicable in the case of monocots. Genes from all classes found in eudicots have been also identified in genomes of such monocots like rice, maize or barley. What's more, it seems that miRNA-mediated regulation of floral organ genes that was observed in the case of Arabidopsis thaliana also takes place in monocots. MiRNA172, miRNA159, miRNA171 and miRNA396 regulate expression of floral organ identity genes in barley, rice and maize, affecting various features of the flower structure, ranging from formation of lemma and palea to the development of reproductive organs. A model of floral development in grasses and its genetic regulation is not yet fully characterized. Further studies on both, the model eudicots and grasses, are needed to unravel this topic. This review provides general overview of genetic model of flower organ identity specification in monocots and it's miRNA-mediated regulation.