Protein phosphatase PP1 regulation of RNA polymerase II transcription termination and allelic exclusion of VSG genes in trypanosomes.

The genomes of Leishmania and trypanosomes are organized into polycistronic transcription units flanked by a modified DNA base J involved in promoting RNA polymerase II (Pol II) termination. We recently characterized a Leishmania complex containing a J-binding protein, PP1 protein phosphatase 1, and PP1 regulatory protein (PNUTS) that controls transcription termination potentially via dephosphorylation of Pol II by PP1. While T. brucei contains eight PP1 isoforms, none purified with the PNUTS complex, complicating the analysis of PP1 function in termination. We now demonstrate that the PP1-binding motif of TbPNUTS is required for function in termination in vivo and that TbPP1-1 modulates Pol II termination in T. brucei and dephosphorylation of the large subunit of Pol II. PP1-1 knock-down results in increased cellular levels of phosphorylated RPB1 accompanied by readthrough transcription and aberrant transcription of the chromosome by Pol II, including Pol I transcribed loci that are typically silent, such as telomeric VSG expression sites involved in antigenic variation. These results provide important insights into the mechanism underlying Pol II transcription termination in primitive eukaryotes that rely on polycistronic transcription and maintain allelic exclusion of VSG genes.

Knockout of protein phosphatase 1 in Leishmania major reveals its role during RNA polymerase II transcription termination.

The genomes of kinetoplastids are organized into polycistronic transcription units that are flanked by a modified DNA base (base J, beta-D-glucosyl-hydroxymethyluracil). Previous work established a role of base J in promoting RNA polymerase II (Pol II) termination in Leishmania major and Trypanosoma brucei. We recently identified a PJW/PP1 complex in Leishmania containing a J-binding protein (JBP3), PP1 phosphatase 1, PP1 interactive-regulatory protein (PNUTS) and Wdr82. Analyses suggested the complex regulates transcription termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of proteins, including Pol II, by PP1. However, we never addressed the role of PP1, the sole catalytic component, in Pol II transcription termination. We now demonstrate that deletion of the PP1 component of the PJW/PP1 complex in L. major, PP1-8e, leads to readthrough transcription at the 3'-end of polycistronic gene arrays. We show PP1-8e has in vitro phosphatase activity that is lost upon mutation of a key catalytic residue and associates with PNUTS via the conserved RVxF motif. Additionally, purified PJW complex with associated PP1-8e, but not complex lacking PP1-8e, led to dephosphorylation of Pol II, suggesting a direct role of PNUTS/PP1 holoenzymes in regulating transcription termination via dephosphorylating Pol II in the nucleus.

Exploring plant cis-regulatory elements at single-cell resolution: overcoming biological and computational challenges to advance plant research.

Cis-regulatory elements (CREs) are important sequences for gene expression and for plant biological processes such as development, evolution, domestication, and stress response. However, studying CREs in plant genomes has been challenging. The totipotent nature of plant cells, coupled with the inability to maintain plant cell types in culture and the inherent technical challenges posed by the cell wall has limited our understanding of how plant cell types acquire and maintain their identities and respond to the environment via CRE usage. Advances in single-cell epigenomics have revolutionized the field of identifying cell-type-specific CREs. These new technologies have the potential to significantly advance our understanding of plant CRE biology, and shed light on how the regulatory genome gives rise to diverse plant phenomena. However, there are significant biological and computational challenges associated with analyzing single-cell epigenomic datasets. In this review, we discuss the historical and foundational underpinnings of plant single-cell research, challenges, and common pitfalls in the analysis of plant single-cell epigenomic data, and highlight biological challenges unique to plants. Additionally, we discuss how the application of single-cell epigenomic data in various contexts stands to transform our understanding of the importance of CREs in plant genomes.

DNAJ protein gene expansion mechanism in Panicoideae and PgDNAJ functional identification in pearl millet.

KEY MESSAGE: Analyze the evolutionary pattern of DNAJ protein genes in the Panicoideae, including pearl millet, to identify and characterize the biological function of PgDNAJ genes in pearl millet. Global warming has become a major factor threatening food security and human development. It is urgent to analyze the heat-tolerant mechanism of plants and cultivate crops that are adapted to high temperature conditions. The Panicoideae are the second largest subfamily of the Poaceae, widely distributed in warm temperate and tropical regions. Many of these species have been reported to have strong adaptability to high temperature stress, such as pearl millet, foxtail millet and sorghum. The evolutionary differences in DNAJ protein genes among 12 Panicoideae species and 10 other species were identified and analyzed. Among them, 79% of Panicoideae DNAJ protein genes were associated with retrotransposon insertion. Analysis of the DNAJ protein pan-gene family in six pearl millet accessions revealed that the non-core genes contained significantly more TEs than the core genes. By identifying and analyzing the distribution and types of TEs near the DNAJ protein genes, it was found that the insertion of Copia and Gypsy retrotransposons provided the source of expansion for the DNAJ protein genes in the Panicoideae. Based on the analysis of the evolutionary pattern of DNAJ protein genes in Panicoideae, the PgDNAJ was obtained from pearl millet through identification. PgDNAJ reduces the accumulation of reactive oxygen species caused by high temperature by activating ascorbate peroxidase (APX), thereby improving the heat resistance of plants. In summary, these data provide new ideas for mining potential heat-tolerant genes in Panicoideae, and help to improve the heat tolerance of other crops.

Diagnosis and cardiac transplantation of a Carney syndrome-induced cardiac myxoma combined with dilated cardiomyopathy: a case report.

BACKGROUND: Carney syndrome is an uncommon autosomal disorder closely linked to mutations in the PRKAR1A gene. Skin lesions are the most pronounced feature of Carney syndrome, affecting over 80% of individuals with this condition. This syndrome is characterized by a triad of myxomas, skin pigmentation, and endocrine hyperfunction, featuring multiple endocrine neoplasms with skin and cardiac involvement. Dilated cardiomyopathy, a primary cardiomyopathy, is defined as the dilation and impaired systolic function of the left or both ventricles. Its clinical presentation varies from being asymptomatic to heart failure or sudden cardiac death, making it a leading global cause of heart failure. Currently, Dilated cardiomyopathy has an estimated prevalence of 1/2500-1/250 individuals, predominantly affecting those aged 30-40 years, with a male-to-female ratio of 3:1. This case report describes a heart failure patient with cardiac myxoma caused by Carney syndrome combined with dilated cardiomyopathy. The patient was successfully treated for heart failure by heart transplantation. CASE PRESENTATION: Herein, we report a case of heart failure due to Carney syndrome that resulted in cardiac myxoma combined with dilated cardiomyopathy. A 35-year-old male was admitted to the hospital three years ago because of sudden chest tightness and shortness of breath. Echocardiography indicated myxoma, and a combination of genetic screening and physical examination confirmed Carney syndrome with cardiac myxoma. Following symptomatic management, he was discharged. Surgical interventions were not considered at the time. However, the patient's chest tightness and shortness of breath symptoms worsened, and he returned to the hospital. A New York Heart Association grade IV heart function was confirmed, and echocardiography indicated the presence of dilated cardiomyopathy accompanied by cardiac myxoma. Ultimately, the patient's heart failure was successfully treated with heart transplantation. CONCLUSIONS: Cardiac myxoma caused by Carney syndrome combined with heart failure caused by dilated cardiomyopathy can be resolved by heart transplantation.

Combined Genome-Wide Association Study and Transcriptome Analysis Reveal Candidate Genes for Resistance to Rust (Puccinia graminis) in Dactylis glomerata.

Rust disease is a common plant disease that can cause wilting, slow growth of plant leaves, and even affect the growth and development of plants. Orchardgrass (Dactylis glomerata L.) is native to temperate regions of Europe, which has been introduced as a superior forage grass in temperate regions worldwide. Orchardgrass has rich genetic diversity and is widely distributed in the world, which may contain rust resistance genes not found in other crops. Therefore, we collected a total of 333 orchardgrass accessions from different regions around the world. Through a genome-wide association study (GWAS) analysis conducted in four different environments, 91 genes that overlap or are adjacent to significant single nucleotide polymorphisms (SNPs) were identified as potential rust disease resistance genes. Combining transcriptome data from susceptible (PI292589) and resistant (PI251814) accessions, the GWAS candidate gene DG5C04160.1 encoding glutathione S-transferase (GST) was found to be important for orchardgrass rust (Puccinia graminis) resistance. Interestingly, by comparing the number of GST gene family members in seven species, it was found that orchardgrass has the most GST gene family members, containing 119 GST genes. Among them, 23 GST genes showed significant differential expression after inoculation with the rust pathogen in resistant and susceptible accessions; 82% of the genes still showed significantly increased expression 14 days after inoculation in resistant accessions, while the expression level significantly decreased in susceptible accessions. These results indicate that GST genes play an important role in orchardgrass resistance to rust (P. graminis) stress by encoding GST to reduce its oxidative stress response.

Milletdb: a multi-omics database to accelerate the research of functional genomics and molecular breeding of millets.

Millets are a class of nutrient-rich coarse cereals with high resistance to abiotic stress; thus, they guarantee food security for people living in areas with extreme climatic conditions and provide stress-related genetic resources for other crops. However, no platform is available to provide a comprehensive and systematic multi-omics analysis for millets, which seriously hinders the mining of stress-related genes and the molecular breeding of millets. Here, a free, web-accessible, user-friendly millets multi-omics database platform (Milletdb, http://milletdb.novogene.com) has been developed. The Milletdb contains six millets and their one related species genomes, graph-based pan-genomics of pearl millet, and stress-related multi-omics data, which enable Milletdb to be the most complete millets multi-omics database available. We stored GWAS (genome-wide association study) results of 20 yield-related trait data obtained under three environmental conditions [field (no stress), early drought and late drought] for 2 years in the database, allowing users to identify stress-related genes that support yield improvement. Milletdb can simplify the functional genomics analysis of millets by providing users with 20 different tools (e.g., 'Gene mapping', 'Co-expression', 'KEGG/GO Enrichment' analysis, etc.). On the Milletdb platform, a gene PMA1G03779.1 was identified through 'GWAS', which has the potential to modulate yield and respond to different environmental stresses. Using the tools provided by Milletdb, we found that the stress-related PLATZs TFs (transcription factors) family expands in 87.5% of millet accessions and contributes to vegetative growth and abiotic stress responses. Milletdb can effectively serve researchers in the mining of key genes, genome editing and molecular breeding of millets.

DNA hypermethylation promotes the flowering of orchardgrass during vernalization.

Vernalization, influenced by environmental factors, is an essential process associated with the productivity of temperate crops, during which epigenetic regulation of gene expression plays an important role. Although DNA methylation is one of the major epigenetic mechanisms associated with the control of gene expression, global changes in DNA methylation in the regulation of gene expression during vernalization-induced flowering of temperate plants remain largely undetermined. To characterize vernalization-associated DNA methylation dynamics, we performed whole-genome bisulfite-treated sequencing and transcriptome sequencing in orchardgrass (Dactylis glomerata) during vernalization. The results revealed that increased levels of genome DNA methylation during the early vernalization of orchardgrass were associated with transcriptional changes in DNA methyltransferase and demethylase genes. Upregulated expression of vernalization-related genes during early vernalization was attributable to an increase in mCHH in the promoter regions of these genes. Application of an exogenous DNA methylation accelerator or overexpression of orchardgrass NUCLEAR POLY(A) POLYMERASE (DgPAPS4) promoted earlier flowering, indicating that DNA hypermethylation plays an important role in vernalization-induced flowering. Collectively, our findings revealed that vernalization-induced hypermethylation is responsible for floral primordium initiation and development. These observations provide a theoretical foundation for further studies on the molecular mechanisms underlying the control of vernalization in temperate grasses.

Carbon dots as specific fluorescent sensors for Hg2+ and glutathione imaging.

Nitrogen-doped carbon dots (NCDs) have been constructed in which coal washing wastewater is used as carbon precursor, tryptophan is added for nitrogen doping and surface functional together with polyethylene glycol. The nitrogen doping and surface functional with electron rich groups resulted in excellent fluorescent properties regarding stability, reversibility, printability with high quantum yield which not only enable the NCDs as fluorescent ink for advanced message encryption, but also realize specific on-off-on fluorescent sensing of Hg2+ and GSH as solution, hydrogel and filter paper sensors. The NCDs had a linear range of 0.01-100 µM and a detection limit of 6.27 nM (RSD 0.33%) for Hg2+ and the NCDs@Hg2+ had a linear range of 0.01-60 µM and a detection limit of 3.53 nM (RSD 1.53%) for GSH in sensing studies with aqueous solutions. In addition, with the low cytotoxicity and good biocompatibility NCDs have been successfully used for imaging Hg2+ and GSH in living MG-63 cells. The presented NCDs recycle waste coal washing water into worthwhile material which can be implemented as promising anti-counterfeiting and message encryption candidates as well as effective Hg2+ and GSH sensing, tracking and removing tools in complicated environmental and biological systems.

Identification of new marker genes from plant single-cell RNA-seq data using interpretable machine learning methods.

An essential step in the analysis of single-cell RNA sequencing data is to classify cells into specific cell types using marker genes. In this study, we have developed a machine learning pipeline called single-cell predictive marker (SPmarker) to identify novel cell-type marker genes in the Arabidopsis root. Unlike traditional approaches, our method uses interpretable machine learning models to select marker genes. We have demonstrated that our method can: assign cell types based on cells that were labelled using published methods; project cell types identified by trajectory analysis from one data set to other data sets; and assign cell types based on internal GFP markers. Using SPmarker, we have identified hundreds of new marker genes that were not identified before. As compared to known marker genes, the new marker genes have more orthologous genes identifiable in the corresponding rice single-cell clusters. The new root hair marker genes also include 172 genes with orthologs expressed in root hair cells in five non-Arabidopsis species, which expands the number of marker genes for this cell type by 35-154%. Our results represent a new approach to identifying cell-type marker genes from scRNA-seq data and pave the way for cross-species mapping of scRNA-seq data in plants.

Mechanistic insights into the amelioration effects of lipopolysaccharide-induced acute lung injury by baicalein: An integrated systems pharmacology study and experimental validation.

BACKGROUND: Acute lung injury is an acute progressive respiratory failure caused by several of non-cardiogenic factors which involves in excessive amplification or uncontrolled inflammatory response. OBJECTIVES: In this study, we investigated the protective effect of baicalein against acute lung injury induced by LPS and explored the underlying mechanisms. METHODS: Forty-eight SPF male C57BL/6 mice were randomly divided into normal group, model group, dexamethasone group and baicalein low-dose, medium-dose and high-dose groups. After 5 days of adaptive feeding, the mice were intraperitoneally injected with LPS and dissected after 12 h. Hematoxylin-eosin staining, ELISA assay, immunofluorescence assay and Western-Blot were applied to appraise microstructural changes and protein expressions of lung tissues. Systems pharmacology study was used to evaluate the protection of baicalein on acute lung injury. FINDINGS: The results showed that baicalein administration could significantly inhibit LPS-induced lung morphological changes, inhibit inflammatory response and pyroptosis. A total of forty-three potential targets of baicalein and acute lung injury were obtained. And PI3K-Akt, TNF and NF-κB were mainly signaling pathways. It is worth mentioning that this experiment also confirmed that NLRP3, caspase-1 and other inflammasome are involved in pyroptosis. CONCLUSION: Baicalein has protected against LPS-induced lung tissues injury via inhibiting inflammatory response and pyroptosis.

The 'Tommy Atkins' mango genome reveals candidate genes for fruit quality.

BACKGROUND: Mango, Mangifera indica L., an important tropical fruit crop, is grown for its sweet and aromatic fruits. Past improvement of this species has predominantly relied on chance seedlings derived from over 1000 cultivars in the Indian sub-continent with a large variation for fruit size, yield, biotic and abiotic stress resistance, and fruit quality among other traits. Historically, mango has been an orphan crop with very limited molecular information. Only recently have molecular and genomics-based analyses enabled the creation of linkage maps, transcriptomes, and diversity analysis of large collections. Additionally, the combined analysis of genomic and phenotypic information is poised to improve mango breeding efficiency. RESULTS: This study sequenced, de novo assembled, analyzed, and annotated the genome of the monoembryonic mango cultivar 'Tommy Atkins'. The draft genome sequence was generated using NRGene de-novo Magic on high molecular weight DNA of 'Tommy Atkins', supplemented by 10X Genomics long read sequencing to improve the initial assembly. A hybrid population between 'Tommy Atkins' x 'Kensington Pride' was used to generate phased haplotype chromosomes and a highly resolved phased SNP map. The final 'Tommy Atkins' genome assembly was a consensus sequence that included 20 pseudomolecules representing the 20 chromosomes of mango and included ~ 86% of the ~ 439 Mb haploid mango genome. Skim sequencing identified ~ 3.3 M SNPs using the 'Tommy Atkins' x 'Kensington Pride' mapping population. Repeat masking identified 26,616 genes with a median length of 3348 bp. A whole genome duplication analysis revealed an ancestral 65 MYA polyploidization event shared with Anacardium occidentale. Two regions, one on LG4 and one on LG7 containing 28 candidate genes, were associated with the commercially important fruit size characteristic in the mapping population. CONCLUSIONS: The availability of the complete 'Tommy Atkins' mango genome will aid global initiatives to study mango genetics.

DeepTE: a computational method for de novo classification of transposons with convolutional neural network.

MOTIVATION: Transposable elements (TEs) classification is an essential step to decode their roles in genome evolution. With a large number of genomes from non-model species becoming available, accurate and efficient TE classification has emerged as a new challenge in genomic sequence analysis. RESULTS: We developed a novel tool, DeepTE, which classifies unknown TEs using convolutional neural networks (CNNs). DeepTE transferred sequences into input vectors based on k-mer counts. A tree structured classification process was used where eight models were trained to classify TEs into super families and orders. DeepTE also detected domains inside TEs to correct false classification. An additional model was trained to distinguish between non-TEs and TEs in plants. Given unclassified TEs of different species, DeepTE can classify TEs into seven orders, which include 15, 24 and 16 super families in plants, metazoans and fungi, respectively. In several benchmarking tests, DeepTE outperformed other existing tools for TE classification. In conclusion, DeepTE successfully leverages CNN for TE classification, and can be used to precisely classify TEs in newly sequenced eukaryotic genomes. AVAILABILITY AND IMPLEMENTATION: DeepTE is accessible at https://github.com/LiLabAtVT/DeepTE. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transcriptome analysis of heat stress and drought stress in pearl millet based on Pacbio full-length transcriptome sequencing.

BACKGROUND: Heat and drought are serious threats for crop growth and development. As the sixth largest cereal crop in the world, pearl millet can not only be used for food and forage but also as a source of bioenergy. Pearl millet is highly tolerant to heat and drought. Given this, it is considered an ideal crop to study plant stress tolerance and can be used to identify heat-resistant genes. RESULTS: In this study, we used Pacbio sequencing data as a reference sequence to analyze the Illumina data of pearl millet that had been subjected to heat and drought stress for 48 h. By summarizing previous studies, we found 26,299 new genes and 63,090 new transcripts, and the number of gene annotations increased by 20.18%. We identified 2792 transcription factors and 1223 transcriptional regulators. There were 318 TFs and 149 TRs differentially expressed under heat stress, and 315 TFs and 128 TRs were differentially expressed under drought stress. We used RNA sequencing to identify 6920 genes and 6484 genes differentially expressed under heat stress and drought stress, respectively. CONCLUSIONS: Through Pacbio sequencing, we have identified more new genes and new transcripts. On the other hand, comparing the differentially expressed genes under heat tolerance with the DEGs under drought stress, we found that even in the same pathway, pearl millet responds with a different protein.

Genome assembly provides insights into the genome evolution and flowering regulation of orchardgrass.

Orchardgrass (Dactylis glomerata L.) is an important forage grass for cultivating livestock worldwide. Here, we report an ~1.84-Gb chromosome-scale diploid genome assembly of orchardgrass, with a contig N50 of 0.93 Mb, a scaffold N50 of 6.08 Mb and a super-scaffold N50 of 252.52 Mb, which is the first chromosome-scale assembled genome of a cool-season forage grass. The genome includes 40 088 protein-coding genes, and 69% of the assembled sequences are transposable elements, with long terminal repeats (LTRs) being the most abundant. The LTRretrotransposons may have been activated and expanded in the grass genome in response to environmental changes during the Pleistocene between 0 and 1 million years ago. Phylogenetic analysis reveals that orchardgrass diverged after rice but before three Triticeae species, and evolutionarily conserved chromosomes were detected by analysing ancient chromosome rearrangements in these grass species. We also resequenced the whole genome of 76 orchardgrass accessions and found that germplasm from Northern Europe and East Asia clustered together, likely due to the exchange of plants along the 'Silk Road' or other ancient trade routes connecting the East and West. Last, a combined transcriptome, quantitative genetic and bulk segregant analysis provided insights into the genetic network regulating flowering time in orchardgrass and revealed four main candidate genes controlling this trait. This chromosome-scale genome and the online database of orchardgrass developed here will facilitate the discovery of genes controlling agronomically important traits, stimulate genetic improvement of and functional genetic research on orchardgrass and provide comparative genetic resources for other forage grasses.

Identification and Distribution of NBS-Encoding Resistance Genes of Dactylis glomerata L. and Its Expression Under Abiotic and Biotic Stress.

Orchardgrass (Dactylis glomerata L.) is drought resistant and tolerant to barren landscapes, making it one of the most important forages for animal husbandry, as well as ecological restoration of rocky landscapes that are undergoing desertification. However, orchardgrass is susceptible to rust, which can significantly reduce its yield and quality. Therefore, understanding the genes that underlie resistance against rust in orchardgrass is critical. The evolution, cloning of plant disease resistance genes, and the analysis of pathogenic bacteria induced expression patterns are important contents in the study of interaction between microorganisms and plants. Genes with nucleotide binding site (NBS) structure are disease-resistant genes ubiquitous in plants and play an important role in plant attacks against various pathogens. Using sequence analysis and re-annotation, we identified 413 NBS resistance genes in orchardgrass. Similar to previous studies, NBS resistance genes containing TIR (toll/interleukin-1 receptor) domain were not found in orchardgrass. The NBS resistance genes can be divided into four types: NBS (up to 264 homologous genes, accounting for 64% of the total number of NBS genes in orchardgrass), NBS-LRR, CC-NBS, and CC-NBS-LRR (minimum of 26 homologous genes, only 6% of the total number of NBS genes in orchardgrass). These 413 NBS resistance genes were unevenly distributed across seven chromosomes where chromosome 5 had up to 99 NBS resistance genes. There were 224 (54%) NBS resistance genes expressed in different tissues (roots, stems, leaves, flowers, and spikes), and we did not detect expression for 45 genes (11%). The remaining 145 (35%) were expressed in some tissues. And we found that 11 NBS resistance genes were differentially expressed under waterlogging stress, 5 NBS resistance genes were differentially expressed under waterlogging and drought stress, and 1 NBS resistance was is differentially expressed under waterlogging and heat stress. Most importantly, we found that 65 NBS resistance genes were significantly expressed in different control groups. On the 7th day of inoculation, 23 NBS resistance genes were differentially expressed in high resistance materials alone, of which 7 NBS resistance genes regulate the "plant-pathogen interaction" pathway by encoding RPM1. At the same time, 2 NBS resistance genes that were differentially expressed in the high resistance material after inoculation were also differentially expressed in abiotic stress. In summary, the NBS resistance gene plays a crucial role in the resistance of orchardgrass to rust.

Autopolyploidization in switchgrass alters phenotype and flowering time via epigenetic and transcription regulation.

Polyploidization is a significant source of genomic and organism diversification during plant evolution, and leads to substantial alterations in plant phenotypes and natural fitness. To help understand the phenotypic and molecular impacts of autopolyploidization, we conducted epigenetic and full-transcriptomic analyses of a synthesized autopolyploid accession of switchgrass (Panicum virgatum) in order to interpret the molecular and phenotypic changes. We found that mCHH levels were decreased in both genic and transposable element (TE) regions, and that TE methylation near genes was decreased as well. Among 142 differentially expressed genes involved in cell division, cellulose biosynthesis, auxin response, growth, and reproduction processes, 75 of them were modified by 122 differentially methylated regions, 10 miRNAs, and 15 siRNAs. In addition, up-regulated PvTOE1 and suppressed PvFT probably contribute to later flowering time of the autopolyploid. The expression changes were probably associated with modification of nearby methylation sites and siRNAs. We also experimentally demonstrated that expression levels of PvFT and PvTOE1 were regulated by DNA methylation, supporting the link between alterations in methylation induced by polyploidization and the phenotypic changes that were observed. Collectively, our results show epigenetic modifications in synthetic autopolyploid switchgrass for the first time, and support the hypothesis that polyploidization-induced methylation is an important cause of phenotypic alterations and is potentially important for plant evolution and improved fitness.

Integration of small RNAs and transcriptome sequencing uncovers a complex regulatory network during vernalization and heading stages of orchardgrass (Dactylis glomerata L.).

BACKGROUND: Flowering is a critical reproductive process in higher plants. Timing of optimal flowering depends upon the coordination among seasonal environmental cues. For cool season grasses, such as Dactylis glomerata, vernalization induced by low temperature provides competence to initiate flowering after prolonged cold. We combined analyses of the transcriptome and microRNAs (miRNAs) to generate a comprehensive resource for regulatory miRNAs and their target circuits during vernalization and heading stages. RESULTS: A total of 3,846 differentially expressed genes (DEGs) and 69 differentially expressed miRNAs were identified across five flowering stages. The expression of miR395, miR530, miR167, miR396, miR528, novel_42, novel_72, novel_107, and novel_123 demonstrated significant variations during vernalization. These miRNA targeted genes were involved in phytohormones, transmembrane transport, and plant morphogenesis in response to vernalization. The expression patterns of DEGs related to plant hormones, stress responses, energy metabolism, and signal transduction changed significantly in the transition from vegetative to reproductive phases. CONCLUSIONS: Five hub genes, c136110_g1 (BRI1), c131375_g1 (BZR1), c133350_g1 (VRN1), c139830_g1 (VIN3), and c125792_g2 (FT), might play central roles in vernalization response. Our comprehensive analyses have provided a useful platform for investigating consecutive transcriptional and post-transcriptional regulation of critical phases in D. glomerata and provided insights into the genetic engineering of flowering-control in cereal crops.

Combinations of Small RNA, RNA, and Degradome Sequencing Uncovers the Expression Pattern of microRNA⁻mRNA Pairs Adapting to Drought Stress in Leaf and Root of Dactylis glomerata L.

Drought stress is a global problem, and the lack of water is a key factor that leads to agricultural shortages. MicroRNAs play a crucial role in the plant drought stress response; however, the microRNAs and their targets involved in drought response have not been well elucidated. In the present study, we used Illumina platform (https://www.illumina.com/) and combined data from miRNA, RNA, and degradome sequencing to explore the drought- and organ-specific miRNAs in orchardgrass (Dactylis glomerata L.) leaf and root. We aimed to find potential miRNA⁻mRNA regulation patterns responding to drought conditions. In total, 519 (486 conserved and 33 novel) miRNAs were identified, of which, 41 miRNAs had significant differential expression among the comparisons (p < 0.05). We also identified 55,366 unigenes by RNA-Seq, where 12,535 unigenes were differently expressed. Finally, our degradome analysis revealed that 5950 transcripts were targeted by 487 miRNAs. A correlation analysis identified that miRNA ata-miR164c-3p and its target heat shock protein family A (HSP70) member 5 gene comp59407_c0 (BIPE3) may be essential in organ-specific plant drought stress response and/or adaptation in orchardgrass. Additionally, Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses found that "antigen processing and presentation" was the most enriched downregulated pathway in adaptation to drought conditions. Taken together, we explored the genes and miRNAs that may be involved in drought adaptation of orchardgrass and identified how they may be regulated. These results serve as a valuable genetic resource for future studies focusing on how plants adapted to drought conditions.

Reference Gene Selection for Quantitative Real-Time Reverse-Transcriptase PCR in Annual Ryegrass (Lolium multiflorum) Subjected to Various Abiotic Stresses.

To select the most stable reference genes in annual ryegrass (Lolium multiflorum), we studied annual ryegrass leaf tissues exposed to various abiotic stresses by qRT-PCR and selected 11 candidate reference genes, i.e., 18S rRNA, E2, GAPDH, eIF4A, HIS3, SAMDC, TBP-1, Unigene71, Unigene77, Unigene755, and Unigene14912. We then used GeNorm, NormFinder, and BestKeeper to analyze the expression stability of these 11 genes, and used RefFinder to comprehensively rank genes according to stability. Under different stress conditions, the most suitable reference genes for studies of leaf tissues of annual ryegrass were different. The expression of the eIF4A gene was the most stable under drought stress. Under saline-alkali stress, Unigene14912 has the highest expression stability. Under acidic aluminum stress, SAMDC expression stability was highest. Under heavy metal stress, Unigene71 expression had the highest stability. According to the software analyses, Unigene14912, HIS3, and eIF4A were the most suitable for analyses of abiotic stress in tissues of annual ryegrass. GAPDH was the least suitable reference gene. In conclusion, selecting appropriate reference genes under abiotic stress not only improves the accuracy of annual ryegrass gene expression analyses, but also provides a theoretical reference for the development of reference genes in plants of the genus Lolium.