The Evolution, Expression Patterns, and Domestication Selection Analysis of the Annexin Gene Family in the Barley Pan-Genome.

Plant annexins constitute a conserved protein family that plays crucial roles in regulating plant growth and development, as well as in responses to both biotic and abiotic stresses. In this study, a total of 144 annexin genes were identified in the barley pan-genome, comprising 12 reference genomes, including cultivated barley, landraces, and wild barley. Their chromosomal locations, physical-chemical characteristics, gene structures, conserved domains, and subcellular localizations were systematically analyzed to reveal the certain differences between wild and cultivated populations. Through a cis-acting element analysis, co-expression network, and large-scale transcriptome analysis, their involvement in growth, development, and responses to various stressors was highlighted. It is worth noting that HvMOREXann5 is only expressed in pistils and anthers, indicating its crucial role in reproductive development. Based on the resequencing data from 282 barley accessions worldwide, genetic variations in thefamily were investigated, and the results showed that 5 out of the 12 identified HvMOREXanns were affected by selection pressure. Genetic diversity and haplotype frequency showed notable reductions between wild and domesticated barley, suggesting that a genetic bottleneck occurred on the annexin family during the barley domestication process. Finally, qRT-PCR analysis confirmed the up-regulation of HvMOREXann7 under drought stress, along with significant differences between wild accessions and varieties. This study provides some insights into the genome organization and genetic characteristics of the annexin gene family in barley at the pan-genome level, which will contribute to better understanding its evolution and function in barley and other crops.

Melatonin regulates dihydrotestosterone formation via its membrane receptor in the epididymal epithelial cells of sheep.

Both melatonin and androgen, which affect sperm fertility, are the important factors in epididymis of male animal. In the present study, we confirmed that melatonin regulates the formation of dihydrotestosterone (DHT) in sheep epididymides. Here, we investigated the localization and the expression levels of melatonin keys synthases AANAT and HIOMT, membrane receptors MT1 and MT2, and nuclear receptor RORα in sheep epididymides and testes. We also cultured epididymal epithelial cells and treated them with different concentrations of melatonin (10-11-10-7 M) and luzindole (10-5 M) and 4P-PDOT (10-5 M) to investigate whether melatonin is involved in the regulation of DHT formation and whether these effects are mediated through its receptor pathways. The results showed that AANAT, HIOMT, MT1, MT2, and RORα were differentially expressed between sheep epididymides and testes. In addition, melatonin is involved in mediating the formation of DHT in epididymal epithelial cells, and its influence on DHT is at least partially regulated by the melatonin receptor pathway. Our findings showed that melatonin regulates the functions of the testes and epididymides through an autocrine mechanism and regulates the formation of androgen in sheep epididymides via the receptor pathway. These results provide a basis for further exploring the regulatory mechanisms of melatonin in animal reproduction.

The effect of melatonin on sheep endometrial epithelial cell apoptosis through the receptor and non-receptor pathways.

Melatonin potentially regulates the female animal reproductive function, but its regulatory mechanism in the apoptosis of sheep endometrial epithelial cells (SEECs) remains to be elucidated. In the present study, immunofluorescence staining, western blotting, and quantitative real-time polymerase chain reaction were performed to detect the distribution of melatonin receptors (MT1 and MT2) in the uterus of sheep and the effect of melatonin via the receptor and non-receptor pathways on the apoptosis of SEECs in vitro. The results showed that melatonin inhibits the apoptosis of SEECs to varying degrees to regulate the expression of estrogen receptors (ERs) and progesterone receptors (PGR) via its interaction with MT1 and MT2. In addition, the ER antagonist partially relieved the inhibitory effect of melatonin on the apoptosis of SEECs, while the PGR antagonist did not. Thus, melatonin mediates endometrial epithelial apoptosis through the MT receptors and also by regulating estrogen function. This study provides evidence of the regulatory mechanism of melatonin on the physiological function of the sheep uterus.

Effects of lipoteichoic and arachidonic acids on the immune-regulatory mechanism of bovine mammary epithelial cells using multi-omics analysis.

Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows. It mainly utilizes the properties of its pathogenic factor, lipoteichoic acid (LTA), to elicit a host-cell inflammatory response and evade the host-cell immune response. Arachidonic acid (AA) has a regulatory role in the inflammatory response, cell metabolism, and apoptosis. The study aimed to establish a cell model by determining the optimal concentration of LTA and AA for cell induction using the Cell Counting Kit-8 assay and the quantitative polymerase chain reaction of interleukin (IL)-1ß, IL-2, and IL-6. MAC-T cells were planted in 36 10-cm2 culture dishes at a density of 1 × 107 cells per dish. They were treated with LTA for 24 h to constitute the LTA group and with AA for 12 h to constitute the AA group. The cells were pretreated with LTA for 24 h followed by treatment with AA for 12 h to constitute the LTA + AA group. Using proteomic, transcriptomic, and metabolomic analyses, this study determined that LTA can regulate the expression of Actin Related protein 2/3 complex (ARPC)3, ARPC4, Charged Multivesicular Body Protein 3, protein kinase cGMP-dependent, NF-κB Inhibitor Alpha,and other genes to affect cellular metabolism, immune regulation and promote apoptosis. In contrast, AA was observed to regulate the expression of genes such as ARPC3, ARPC4, Charged Multivesicular Body Protein 3, Laminin Gamma 1, Insulin Receptor, Filamin B, and Casein Kinase 1 Epsilon to inhibit cellular apoptosis and promote immune regulation, which provides a theoretical basis for future studies.

Genomic and Phenotypic Divergence in Wild Barley Driven by Microgeographic Adaptation.

Microgeographic adaptation is a fundamental driving force of evolution, but the underlying causes remain undetermined. Here, the phenotypic, genomic and transcriptomic variations of two wild barley populations collected from sharply divergent and adjacent micro-geographic sites to identify candidate genes associated with edaphic local adaptation are investigated. Common garden and reciprocal transplant studies show that large phenotypic differentiation and local adaptation to soils occur between these populations. Genetic, phylogenetic and admixture analyses based on population resequencing show that significant genetic divergences occur between basalt and chalk populations. These divergences are consistent with the phenotypic variations observed in the field. Genome sweep analyses reveal 162.7 Mb of selected regions driven by edaphic local adaptation, in which 445 genes identified, including genes associated with root architecture, metal transport/detoxification, and ABA signaling. When the phenotypic, genomic and transcriptomic data are combined, HvMOR, encoding an LBD transcription factor, is determined to be the vital candidate for regulating the root architecture to adapt to edaphic conditions at the microgeographic scale. This study provides new insights into the genetic basis of edaphic adaptation and demonstrates that edaphic factors may contribute to the evolution and speciation of barley.

microRNA-216a protects against human retinal microvascular endothelial cell injury in diabetic retinopathy by suppressing the NOS2/JAK/STAT axis.

OBJECTIVE: Since microRNAs (miRNAs) represent as effective therapeutic targets for diabetic retinopathy (DR), we identified aberrantly expressed miRNAs related to cellular dysfunction in DR and further detected their potential targets. This study aimed to explore the synergistic effect of miR-216a, inducible nitric oxide synthase 2 (NOS2) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway on human retinal microvascular endothelial cell (HRMEC) injury in DR. METHODS: The differentially expressed genes in DR were obtained by GEO database, and the downstream signaling pathways and upstream targeted miRNAs were obtained through bioinformatics analysis. Subsequently, a DR model rat was established, and the target miR-216a was overexpressed to observe the pathological and morphological changes of the rat retina and the levels of inflammatory factors. Then, HRMECs were extracted and added with d-Glucose, and then transfected with miR-216a, NOS2 or adding JAK/STAT signaling pathway specific inhibitor to observe changes in cell activity and inflammatory damage. RESULTS: NOS2 was significantly upregulated, and the JAK/STAT signaling pathway was significantly activated in DR. miR-216a targeted NOS2, which played a protective role in the retina of DR rats. Moreover, in cell experiments, overexpression of miR-216a promoted the viability of HRMECs under d-glucose treatment, and inhibited NOS2 expression and the JAK/STAT signaling pathway activation. CONCLUSION: This study suggests that miR-216a protects against HRMECs injury in DR by suppressing the NOS2/JAK/STAT axis.

The improved assembly of 7DL chromosome provides insight into the structure and evolution of bread wheat.

Wheat is one of the most important staple crops worldwide and also an excellent model species for crop evolution and polyploidization studies. The breakthrough of sequencing the bread wheat genome and progenitor genomes lays the foundation to decipher the complexity of wheat origin and evolutionary process as well as the genetic consequences of polyploidization. In this study, we sequenced 3286 BACs from chromosome 7DL of bread wheat cv. Chinese Spring and integrated the unmapped contigs from IWGSC v1 and available PacBio sequences to close gaps present in the 7DL assembly. In total, 8043 out of 12 825 gaps, representing 3 491 264 bp, were closed. We then used the improved assembly of 7DL to perform comparative genomic analysis of bread wheat (Ta7DL) and its D donor, Aegilops tauschii (At7DL), to identify domestication signatures. Results showed a strong syntenic relationship between Ta7DL and At7DL, although some small rearrangements were detected at the distal regions. A total of 53 genes appear to be lost genes during wheat polyploidization, with 23% (12 genes) as RGA (disease resistance gene analogue). Furthermore, 86 positively selected genes (PSGs) were identified, considered to be domestication-related candidates. Finally, overlapping of QTLs obtained from GWAS analysis and PSGs indicated that TraesCS7D02G321000 may be one of the domestication genes involved in grain morphology. This study provides comparative information on the sequence, structure and organization between bread wheat and Ae. tauschii from the perspective of the 7DL chromosome, which contribute to better understanding of the evolution of wheat, and supports wheat crop improvement.

Transcriptional Dynamics of Grain Development in Barley (Hordeum vulgare L.).

Grain development, as a vital process in the crop's life cycle, is crucial for determining crop quality and yield. However, the molecular basis and regulatory network of barley grain development is not well understood at present. Here, we investigated the transcriptional dynamics of barley grain development through RNA sequencing at four developmental phases, including early prestorage phase (3 days post anthesis (DPA)), late prestorage or transition phase (8 DPA), early storage phase (13 DPA), and levels off stages (18 DPA). Transcriptome profiling found that pronounced shifts occurred in the abundance of transcripts involved in both primary and secondary metabolism during grain development. The transcripts' activity was decreased during maturation while the largest divergence was observed between the transitions from prestorage phase to storage phase, which coincided with the physiological changes. Furthermore, the transcription factors, hormone signal transduction-related as well as sugar-metabolism-related genes, were found to play a crucial role in barley grain development. Finally, 4771 RNA editing events were identified in these four development stages, and most of the RNA editing genes were preferentially expressed at the prestore stage rather than in the store stage, which was significantly enriched in "essential" genes and plant hormone signal transduction pathway. These results suggested that RNA editing might act as a 'regulator' to control grain development. This study systematically dissected the gene expression atlas of barley grain development through transcriptome analysis, which not only provided the potential targets for further functional studies, but also provided insights into the dynamics of gene regulation underlying grain development in barley and beyond.

Melatonin: A Small Molecule but Important for Salt Stress Tolerance in Plants.

Salt stress is one of the most serious limiting factors in worldwide agricultural production, resulting in huge annual yield loss. Since 1995, melatonin (N-acetyl-5-methoxytryptamine)-an ancient multi-functional molecule in eukaryotes and prokaryotes-has been extensively validated as a regulator of plant growth and development, as well as various stress responses, especially its crucial role in plant salt tolerance. Salt stress and exogenous melatonin lead to an increase in endogenous melatonin levels, partly via the phyto-melatonin receptor CAND2/PMTR1. Melatonin plays important roles, as a free radical scavenger and antioxidant, in the improvement of antioxidant systems under salt stress. These functions improve photosynthesis, ion homeostasis, and activate a series of downstream signals, such as hormones, nitric oxide (NO) and polyamine metabolism. Melatonin also regulates gene expression responses to salt stress. In this study, we review recent literature and summarize the regulatory roles and signaling networks involving melatonin in response to salt stress in plants. We also discuss genes and gene families involved in the melatonin-mediated salt stress tolerance.

Effect of overflow tailings properties on cemented paste backfill.

This paper presents the results of an experimental study on the unconfined compressive strength (UCS), indirect tensile strength (ITS), microstructural properties and environmental impacts of overflow tailings (OFT) cemented paste backfill (CPB). The test results show that the poor graded OFT contain 47.15% particles with size below 20 µm, and high content of oxide and sulfide. The slump of fresh CPB mixture ranges from 19.50 cm to 21.60 cm, and its flowability meets the transport requirements. After being cured for 28 days, the UCS and ITS of CPB are less than 2 MPa and 0.5 MPa, respectively. Meanwhile, CPB exhibited low residual strength and high brittleness. The low strength is mainly due to the use of fine OFT. The results show that sulphate also has negative effect on strength and dissolve calcium silicate hydrate (C-S-H) while promoting the formation of secondary products (gypsum and ettringite), leading to generate cracks and decrease UCS at 28 curing time of some CPB mixtures. Increasing the cement content and solid mass concentration is the effective methods to improve the mechanical properties of CPB. The nuclear magnetic resonance (NMR) tests show that fine OFT with low osmotic coefficient (3.10e-06 cm/s) and high sulfur content cause high porosity from 25.0% to 33.1% of CPB, and the increase of the porosity is observed a negative influence on the UCS, but no obvious relationship between ITS and porosity is obtained. Scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS) tests show that OFT particles were covered by C-S-H gel which can enhance the compactness and integrity of CPB. The SEM tests also found secondary products gypsum and ettringite. These finding suggest that OFT can be suitably used as backfill material to fill the underground stopes.

Genome-wide sequence and expressional analysis of autophagy Gene family in bread wheat (Triticum aestivum L.).

Autophagy, a highly conserved intracellular degradation system, is regarded to be responsible for self-defense and protect cells from abiotic stress. Extensive studies have demonstrated that autophagy plays a crucial role in regulating plant growth and development as well as in response to diverse stresses. However, little is known about autophagy-associated genes (ATGs) in wheat, especially those involved in the regulatory network of stress processes. In this study, a total of 108 putative wheat ATGs (TaATG) were obtained based on a genome-wide search approach. Phylogenetic analysis classified them into 13 subfamilies, of which the TaAtg16 subfamily consisted of 29 members, ranking it the largest subfamily. The conserved motif compositions as well as their exon-intron structures were systematically analyzed and strongly supported the classification. The homoeologous genes tended to have similar gene features during wheat polyploidization. Furthermore, a total of 114 putative cis-elements were found, and those related to hormone, stress, and light responsiveness were abundantly presented in the promoter regions. Co-expression network analysis revealed that orthologous VAMP727 was the hub node of the whole network, and complex interactions were also found. Finally, the expression profiles of TaATGs among different tissues and under abiotic stresses were investigated to identify tissue-specific or stress-responsive candidates, and then 14 were validated by wet-lab analysis. Results showed that the TaAtg8 subfamily played a crucial role in tissue autophagy and stress defense, which could be considered as processes that are candidates for further functional study. This was the first study to comprehensively investigate the ATG family in wheat, which ultimately provided important clues for further functional analysis and also took a step toward uncovering the evolutionary mechanism of ATG genes in wheat and beyond.

Genome-Wide Identification and Expression Analysis of the HD-Zip Gene Family in Wheat (Triticum aestivum L.).

The homeodomain-leucine zipper (HD-Zip) gene family, as plant-specific transcription factors, plays an important role in plant development and growth as well as in the response to diverse stresses. Although HD-Zip genes have been extensively studied in many plants, they had not yet been studied in wheat, especially those involved in response to abiotic stresses. In this study, 46 wheat HD-Zip genes were identified using a genome-wide search method. Phylogenetic analysis classified these genes into four groups, numbered 4, 5, 17 and 20 respectively. In total, only three genes with A, B and D homoeologous copies were identified. Furthermore, the gene interaction networks found that the TaHDZ genes played a critical role in the regulatory pathway of organ development and osmotic stress. Finally, the expression profiles of the wheat HD-Zips in different tissues and under various abiotic stresses were investigated using the available RNA sequencing (RNA-Seq) data and then validated by quantitative real-time polymerase chain reaction (qRT-PCR) to obtain the tissue-specific and stress-responsive candidates. This study systematically identifies the HD-Zip gene family in wheat at the genome-wide level, providing important candidates for further functional analysis and contributing to the better understanding of the molecular basis of development and stress tolerance in wheat.

De novo Assembly and Characterization of the Transcriptome of Broomcorn Millet (Panicum miliaceum L.) for Gene Discovery and Marker Development.

Broomcorn millet (Panicum miliaceum L.) is one of the world's oldest cultivated cereals, which is well-adapted to extreme environments such as drought, heat, and salinity with an efficient C4 carbon fixation. Discovery and identification of genes involved in these processes will provide valuable information to improve the crop for meeting the challenge of global climate change. However, the lack of genetic resources and genomic information make gene discovery and molecular mechanism studies very difficult. Here, we sequenced and assembled the transcriptome of broomcorn millet using Illumina sequencing technology. After sequencing, a total of 45,406,730 and 51,160,820 clean paired-end reads were obtained for two genotypes Yumi No. 2 and Yumi No. 3. These reads were mixed and then assembled into 113,643 unigenes, with the length ranging from 351 to 15,691 bp, of which 62,543 contings could be assigned to 315 gene ontology (GO) categories. Cluster of orthologous groups and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses assigned could map 15,514 unigenes into 202 KEGG pathways and 51,020 unigenes to 25 COG categories, respectively. Furthermore, 35,216 simple sequence repeats (SSRs) were identified in 27,055 unigene sequences, of which trinucleotides were the most abundant repeat unit, accounting for 66.72% of SSRs. In addition, 292 differentially expressed genes were identified between the two genotypes, which were significantly enriched in 88 GO terms and 12 KEGG pathways. Finally, the expression patterns of four selected transcripts were validated through quantitative reverse transcription polymerase chain reaction analysis. Our study for the first time sequenced and assembled the transcriptome of broomcorn millet, which not only provided a rich sequence resource for gene discovery and marker development in this important crop, but will also facilitate the further investigation of the molecular mechanism of its favored agronomic traits and beyond.

Transcriptome-wide identification and expression profiles of the WRKY transcription factor family in Broomcorn millet (Panicum miliaceum L.).

BACKGROUND: WRKY genes, as the most pivotal transcription factors in plants, play the indispensable roles in regulating various physiological processes, including plant growth and development as well as in response to stresses. Broomcorn millet is one of the most important crops in drought areas worldwide. However, the WRKY gene family in broomcorn millet remains unknown. RESULTS: A total of 32 PmWRKY genes were identified in this study using computational prediction method. Structural analysis found that PmWRKY proteins contained a highly conserved motif WRKYGQK and two common variant motifs, namely WRKYGKK and WRKYGEK. Phylogenetic analysis of PmWRKYs together with the homologous genes from the representative species could classify them into three groups, with the number of 1, 15, and 16, respectively. Finally, the transcriptional profiles of these 32 PmWRKY genes in various tissues or under different abiotic stresses were systematically investigated using qRT-PCR analysis. Results showed that the expression level of 22 PmWRKY genes varied significantly under one or more abiotic stress treatments, which could be defined as abiotic stress-responsive genes. CONCLUSIONS: This was the first study to identify the organization and transcriptional profiles of PmWRKY genes, which not only facilitates the functional analysis of the PmWRKY genes, and also lays the foundation to reveal the molecular mechanism of stress tolerance in this important crop.

Characterization of microRNAs and their targets in wild barley (Hordeum vulgare subsp. spontaneum) using deep sequencing.

MicroRNAs (miRNA) are a class of small, endogenous RNAs that play a negative regulatory role in various developmental and metabolic processes of plants. Wild barley (Hordeum vulgare subsp. spontaneum), as the progenitor of cultivated barley (Hordeum vulgare subsp. vulgare), has served as a valuable germplasm resource for barley genetic improvement. To survey miRNAs in wild barley, we sequenced the small RNA library prepared from wild barley using the Illumina deep sequencing technology. A total of 70 known miRNAs and 18 putative novel miRNAs were identified. Sequence analysis revealed that all of the miRNAs identified in wild barley contained the highly conserved hairpin sequences found in barley cultivars. MiRNA target predictions showed that 12 out of 52 miRNA families were predicted to target transcription factors, including 8 highly conserved miRNA families in plants and 4 wheat-barley conserved miRNA families. In addition to transcription factors, other predicted target genes were involved in diverse physiological and metabolic processes and stress defense. Our study for the first time reported the large-scale investigation of small RNAs in wild barley, which will provide essential information for understanding the regulatory role of miRNAs in wild barley and also shed light on future practical utilization of miRNAs for barley improvement.

Global Identification of MicroRNAs and Their Targets in Barley under Salinity Stress.

Salinity is a major limiting factor for agricultural production worldwide. A better understanding of the mechanisms of salinity stress response will aid efforts to improve plant salt tolerance. In this study, a combination of small RNA and mRNA degradome sequencing was used to identify salinity responsive-miRNAs and their targets in barley. A total of 152 miRNAs belonging to 126 families were identified, of which 44 were found to be salinity responsive with 30 up-regulated and 25 down-regulated respectively. The majority of the salinity-responsive miRNAs were up-regulated at the 8h time point, while down-regulated at the 3h and 27h time points. The targets of these miRNAs were further detected by degradome sequencing coupled with bioinformatics prediction. Finally, qRT-PCR was used to validate the identified miRNA and their targets. Our study systematically investigated the expression profile of miRNA and their targets in barley during salinity stress phase, which can contribute to understanding how miRNAs respond to salinity stress in barley and other cereal crops.

Complete chloroplast genome sequence of a major invasive species, crofton weed (Ageratina adenophora).

BACKGROUND: Crofton weed (Ageratina adenophora) is one of the most hazardous invasive plant species, which causes serious economic losses and environmental damages worldwide. However, the sequence resource and genome information of A. adenophora are rather limited, making phylogenetic identification and evolutionary studies very difficult. Here, we report the complete sequence of the A. adenophora chloroplast (cp) genome based on Illumina sequencing. METHODOLOGY/PRINCIPAL FINDINGS: The A. adenophora cp genome is 150, 689 bp in length including a small single-copy (SSC) region of 18, 358 bp and a large single-copy (LSC) region of 84, 815 bp separated by a pair of inverted repeats (IRs) of 23, 755 bp. The genome contains 130 unique genes and 18 duplicated in the IR regions, with the gene content and organization similar to other Asteraceae cp genomes. Comparative analysis identified five DNA regions (ndhD-ccsA, psbI-trnS, ndhF-ycf1, ndhI-ndhG and atpA-trnR) containing parsimony-informative characters higher than 2%, which may be potential informative markers for barcoding and phylogenetic analysis. Repeat structure, codon usage and contraction of the IR were also investigated to reveal the pattern of evolution. Phylogenetic analysis demonstrated a sister relationship between A. adenophora and Guizotia abyssinica and supported a monophyly of the Asterales. CONCLUSION: We have assembled and analyzed the chloroplast genome of A. adenophora in this study, which was the first sequenced plastome in the Eupatorieae tribe. The complete chloroplast genome information is useful for plant phylogenetic and evolutionary studies within this invasive species and also within the Asteraceae family.

Identification and characterization of microRNAs from barley (Hordeum vulgare L.) by high-throughput sequencing.

MicroRNAs (miRNAs) are a class of endogenous RNAs that regulates the gene expression involved in various biological and metabolic processes. Barley is one of the most important cereal crops worldwide and is a model organism for genetic and genomic studies in Triticeae species. However, the miRNA research in barley has lagged behind other model species in grass family. To obtain more information of miRNA genes in barley, we sequenced a small RNA library created from a pool of equal amounts of RNA from four different tissues using Solexa sequencing. In addition to 126 conserved miRNAs (58 families), 133 novel miRNAs belonging to 50 families were identified from this sequence data set. The miRNA* sequences of 15 novel miRNAs were also discovered, suggesting the additional evidence for existence of these miRNAs. qRT-PCR was used to examine the expression pattern of six randomly selected miRNAs. Some miRNAs involved in drought and salt stress response were also identified. Furthermore, the potential targets of these putative miRNAs were predicted using the psRNATarget tools. Our results significantly increased the number of novel miRNAs in barley, which should be useful for further investigation into the biological functions and evolution of miRNAs in barley and other species.