REVEILLE2 thermosensitive splicing: a molecular basis for the integration of nocturnal temperature information by the Arabidopsis circadian clock.

Cold stress is one of the major environmental factors that limit growth and yield of plants. However, it is still not fully understood how plants account for daily temperature fluctuations, nor how these temperature changes are integrated with other regulatory systems such as the circadian clock. We demonstrate that REVEILLE2 undergoes alternative splicing after chilling that increases accumulation of a transcript isoform encoding a MYB-like transcription factor. We explore the biological function of REVEILLE2 in Arabidopsis thaliana using a combination of molecular genetics, transcriptomics, and physiology. Disruption of REVEILLE2 alternative splicing alters regulatory gene expression, impairs circadian timing, and improves photosynthetic capacity. Changes in nuclear gene expression are particularly apparent in the initial hours following chilling, with chloroplast gene expression subsequently upregulated. The response of REVEILLE2 to chilling extends our understanding of plants immediate response to cooling. We propose that the circadian component REVEILLE2 restricts plants responses to nocturnal reductions in temperature, thereby enabling appropriate responses to daily environmental changes.

Discrimination of Multidrug Resistance in Cancer Cells Achieved Using Single-Cell Analysis.

The biophysical signatures of single cells, such as multidrug resistance (MDR), may easily change during their various disease states. Therefore, there is an ever-growing need for advanced methods to study and analyze the response of cancer cells to therapeutic intervention. To determine the cancer cells and responses to various cancer therapies, from a cell mortality perspective, we report a label-free and real-time method to monitor the in situ responses of ovarian cancer cells using a single-cell bioanalyzer (SCB). The SCB instrument was used to detect different ovarian cancer cells, such as NCI/ADR-RES cells, which are multidrug resistant (MDR), and non-MDR OVCAR-8 cells. The discrimination of ovarian cells has been achieved at the single-cell level by measuring drug accumulation quantitatively in real time, in which the accumulation is high in non-MDR single cells without drug efflux but is low in MDR single cells which are not efflux-free. The SCB was constructed as an inverted microscope for optical imaging and fluorescent measurement of a single cell that was retained in a microfluidic chip. The single ovarian cancer cell retained in the chip offered sufficient fluorescent signals for the SCB to measure the accumulation of daunorubicin (DNR) in the single cell in the absence of cyclosporine A (CsA). The same cell allows us to detect the enhanced drug accumulation due to MDR modulation in the presence of CsA, which is the MDR inhibitor. The measurement of drug accumulation in a cell was achieved after it was captured in the chip for one hour, with the correction of background interference. The detection of accumulation enhancement due to MDR modulation by CsA was determined in terms of either the accumulation rate or enhanced concentration of DNR in the single cell (same cell, p < 0.01). It showed that with the effectiveness of efflux blocking by CsA, the intracellular DNR concentration in a single cell increased by threefold against its same cell control. This single-cell bioanalyzer instrument has the ability to discriminate MDR in different ovarian cells due to drug efflux in them by eliminating the interference of background fluorescence and by using the same cell control.

ProtView: A Versatile Tool for In Silico Protease Evaluation and Selection in a Proteomic and Proteogenomic Context.

Many tools have been created to generate in silico proteome digests with different protease enzymes and provide useful information for selecting optimal digest schemes for specific needs. This can save on time and resources and generate insights on the observable proteome. However, there remains a need for a tool that evaluates digest schemes beyond protein and amino acid coverages in the proteomic domain. Here, we present ProtView, a versatile in silico protease combination digest evaluation workflow that maps in silico-digested peptides to both protein and genome references, so that the potential observable portions of the proteome, transcriptome, and genome can be identified. The proteomic identification and quantification of evidence for transcriptional, co-transcriptional, post-transcriptional, translational, and post-translational regulation can all be examined in silico with ProtView prior to an experiment. Benchmarking against biological data comparing multiple proteases shows that ProtView can correctly estimate performances among the digest schemes. ProtView provides this information in a way that is easy to interpret, allowing for digest schemes to be evaluated before carrying out an experiment, in context that can optimize both proteomic and proteogenomic experiments. ProtView is available at https://github.com/SSPuliasis/ProtView.

Effects of Graphene Oxide on Plant Growth: A Review.

Several reports of graphene oxide (GO) promoting plant growth have sparked interest in its potential applications in agroforestry. However, there are still some toxicity studies that have raised concerns about the biosafety of GO. These reports show conflicting results from different perspectives, such as plant physiology, biochemistry, cytology, and molecular biology, regarding the beneficial and detrimental effects of GO on plant growth. Seemingly inconsistent studies make it difficult to effectively apply GO in agroforestry. Therefore, it is crucial to review and analyze the current literature on the impacts of GO on plant growth and its physiological parameters. Here, the biological effects of GO on plant growth are summarized. It is proposed that an appropriate concentration of GO may be conducive to its positive effects, and the particle size of GO should be considered when GO is applied in agricultural applications. This review provides a comprehensive understanding of the effects of GO on plant growth to facilitate its safe and effective use.

Second near-infrared fluorescent dye for lateral flow immunoassays rapid detection of influenza A/B virus.

Sensitive and rapid diagnostic point of care testing (POCT) system is of great significance to prevent and control human virus infection. Here reported an immunochromatographic strip technology. The second near-infrared (NIR-II) fluorescent dye encapsulated into polystyrene (PS) nanoparticles, was integrated into a lateral flow assay platform to achieve excellent detection of influenza A/B. This surface-functionalized and mono-dispersed PS nanoparticles has been conjugated with influenza nucleoprotein monoclonal antibody as targets for influenza antigen-detection. This assay achieved the detection limit of 0.015 ng/mL for influenza A nucleoprotein and 4.3*10-5 HAU/mL (102.08 TCID50/mL) influenza A virus (influenza B: 0.037 ng/mL, 9.7*10-7 HAU/mL (100.43 TCID50/mL)). Compared with an Au-based lateral flow test strip, the strip's sensitivity is about 16-fold higher than it. Strip detection properties remain stable for 6 months under 4 °C to 30 °C storage. The assay's intra assay variation is 5.14% and the inter assay variation is 7.74%. Other potential endogenous and exogenous interfering substances (whole blood, nasal mucin, saliva, antipyretics, antihistamines and neuraminidase inhibitors) showed negative results, which verified the excellent specificity of this method. This assay was successfully applied to the POCT quantitative detection of influenza A/B virus, the sensitivity to influenza A and B viruses was 70% and 87.5% respectively, and the specificity was 100%. Therefore, these microspheres can be used as an effective material for rapid POCT detection in clinical specimens.

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis.

BACKGROUND: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. RESULTS: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. CONCLUSIONS: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

BaRTv2: a highly resolved barley reference transcriptome for accurate transcript-specific RNA-seq quantification.

Accurate characterisation of splice junctions (SJs) as well as transcription start and end sites in reference transcriptomes allows precise quantification of transcripts from RNA-seq data, and enables detailed investigations of transcriptional and post-transcriptional regulation. Using novel computational methods and a combination of PacBio Iso-seq and Illumina short-read sequences from 20 diverse tissues and conditions, we generated a comprehensive and highly resolved barley reference transcript dataset from the European 2-row spring barley cultivar Barke (BaRTv2.18). Stringent and thorough filtering was carried out to maintain the quality and accuracy of the SJs and transcript start and end sites. BaRTv2.18 shows increased transcript diversity and completeness compared with an earlier version, BaRTv1.0. The accuracy of transcript level quantification, SJs and transcript start and end sites have been validated extensively using parallel technologies and analysis, including high-resolution reverse transcriptase-polymerase chain reaction and 5'-RACE. BaRTv2.18 contains 39 434 genes and 148 260 transcripts, representing the most comprehensive and resolved reference transcriptome in barley to date. It provides an important and high-quality resource for advanced transcriptomic analyses, including both transcriptional and post-transcriptional regulation, with exceptional resolution and precision.

The value of genotype-specific reference for transcriptome analyses in barley.

It is increasingly apparent that although different genotypes within a species share "core" genes, they also contain variable numbers of "specific" genes and different structures of "core" genes that are only present in a subset of individuals. Using a common reference genome may thus lead to a loss of genotype-specific information in the assembled Reference Transcript Dataset (RTD) and the generation of erroneous, incomplete or misleading transcriptomics analysis results. In this study, we assembled genotype-specific RTD (sRTD) and common reference-based RTD (cRTD) from RNA-seq data of cultivated Barke and Morex barley, respectively. Our quantitative evaluation showed that the sRTD has a significantly higher diversity of transcripts and alternative splicing events, whereas the cRTD missed 40% of transcripts present in the sRTD and it only has ∼70% accurate transcript assemblies. We found that the sRTD is more accurate for transcript quantification as well as differential expression analysis. However, gene-level quantification is less affected, which may be a reasonable compromise when a high-quality genotype-specific reference is not available.

Experimental Design for Time-Series RNA-Seq Analysis of Gene Expression and Alternative Splicing.

RNA-sequencing (RNA-seq) is currently the method of choice for analysis of differential gene expression. To fully exploit the wealth of data generated from genome-wide transcriptomic approaches, the initial design of the experiment is of paramount importance. Biological rhythms in nature are pervasive and are driven by endogenous gene networks collectively known as circadian clocks. Measuring circadian gene expression requires time-course experiments which take into account time-of-day factors influencing variability in expression levels. We describe here an approach for characterizing diurnal changes in expression and alternative splicing for plants undergoing cooling. The method uses inexpensive everyday laboratory equipment and utilizes an RNA-seq application (3D RNA-seq) that can handle complex experimental designs and requires little or no prior bioinformatics expertise.

The proteome of developing barley anthers during meiotic prophase I.

Flowering plants reproduce sexually by combining a haploid male and female gametophyte during fertilization. Male gametophytes are localized in the anthers, each containing reproductive (meiocyte) and non-reproductive tissue necessary for anther development and maturation. Meiosis, where chromosomes pair and exchange their genetic material during a process called recombination, is one of the most important and sensitive stages in breeding, ensuring genetic diversity. Most anther development studies have focused on transcript variation, but very few have been correlated with protein abundance. Taking advantage of a recently published barley anther transcriptomic (BAnTr) dataset and a newly developed sensitive mass spectrometry-based approach to analyse the barley anther proteome, we conducted high-resolution mass spectrometry analysis of barley anthers, collected at six time points and representing their development from pre-meiosis to metaphase. Each time point was carefully staged using immunocytology, providing a robust and accurate staging mirroring our previous BAnTr dataset. We identified >6100 non-redundant proteins including 82 known and putative meiotic proteins. Although the protein abundance was relatively stable throughout prophase I, we were able to quantify the dynamic variation of 336 proteins. We present the first quantitative comparative proteomics study of barley anther development during meiotic prophase I when the important process of homologous recombination is taking place.

Chromatin accessibility landscapes activated by cell-surface and intracellular immune receptors.

Activation of cell-surface and intracellular receptor-mediated immunity results in rapid transcriptional reprogramming that underpins disease resistance. However, the mechanisms by which co-activation of both immune systems lead to transcriptional changes are not clear. Here, we combine RNA-seq and ATAC-seq to define changes in gene expression and chromatin accessibility. Activation of cell-surface or intracellular receptor-mediated immunity, or both, increases chromatin accessibility at induced defence genes. Analysis of ATAC-seq and RNA-seq data combined with publicly available information on transcription factor DNA-binding motifs enabled comparison of individual gene regulatory networks activated by cell-surface or intracellular receptor-mediated immunity, or by both. These results and analyses reveal overlapping and conserved transcriptional regulatory mechanisms between the two immune systems.

Effects of graphene on morphology, microstructure and transcriptomic profiling of Pinus tabuliformis Carr. roots.

Graphene has shown great potential for improving growth of many plants, but its effect on woody plants remains essentially unstudied. In this work, Pinus tabuliformis Carr. bare-rooted seedlings grown outdoors in pots were irrigated with a graphene solution over a concentration range of 0-50 mg/L for six months. Graphene was found to stimulate root growth, with a maximal effect at 25 mg/L. We then investigated root microstructure and carried out transcript profiling of root materials treated with 0 and 25 mg/L graphene. Graphene treatment resulted in plasma-wall separation and destruction of membrane integrity in root cells. More than 50 thousand of differentially expressed genes (DEGs) were obtained by RNA sequencing, among which 6477 could be annotated using other plant databases. The GO enrichment analysis and KEGG pathway analysis of the annotated DEGs indicated that abiotic stress responses, which resemble salt stress, were induced by graphene treatment in roots, while responses to biotic stimuli were inhibited. Numerous metabolic processes and hormone signal transduction pathways were altered by the treatment. The growth promotion effects of graphene may be mediated by encouraging proline synthesis, and suppression of the expression of the auxin response gene SMALL AUXIN UP-REGULATED RNA 41 (SAUR41), PYL genes which encode ABA receptors, and GSK3 homologs.

3D RNA-seq: a powerful and flexible tool for rapid and accurate differential expression and alternative splicing analysis of RNA-seq data for biologists.

RNA-sequencing (RNA-seq) analysis of gene expression and alternative splicing should be routine and robust but is often a bottleneck for biologists because of different and complex analysis programs and reliance on specialized bioinformatics skills. We have developed the '3D RNA-seq' App, an R shiny App and web-based pipeline for the comprehensive analysis of RNA-seq data from any organism. It represents an easy-to-use, flexible and powerful tool for analysis of both gene and transcript-level gene expression to identify differential gene/transcript expression, differential alternative splicing and differential transcript usage (3D) as well as isoform switching from RNA-seq data. 3D RNA-seq integrates state-of-the-art differential expression analysis tools and adopts best practice for RNA-seq analysis. The program is designed to be run by biologists with minimal bioinformatics experience (or by bioinformaticians) allowing lab scientists to analyse their RNA-seq data. It achieves this by operating through a user-friendly graphical interface which automates the data flow through the programs in the pipeline. The comprehensive analysis performed by 3D RNA-seq is extremely rapid and accurate, can handle complex experimental designs, allows user setting of statistical parameters, visualizes the results through graphics and tables, and generates publication quality figures such as heat-maps, expression profiles and GO enrichment plots. The utility of 3D RNA-seq is illustrated by analysis of data from a time-series of cold-treated Arabidopsis plants and from dexamethasone-treated male and female mouse cortex and hypothalamus data identifying dexamethasone-induced sex- and brain region-specific differential gene expression and alternative splicing.

Differential nucleosome occupancy modulates alternative splicing in Arabidopsis thaliana.

Alternative splicing (AS) is a major gene regulatory mechanism in plants. Recent evidence supports co-transcriptional splicing in plants, hence the chromatin state can impact AS. However, how dynamic changes in the chromatin state such as nucleosome occupancy influence the cold-induced AS remains poorly understood. Here, we generated transcriptome (RNA-Seq) and nucleosome positioning (MNase-Seq) data for Arabidopsis thaliana to understand how nucleosome positioning modulates cold-induced AS. Our results show that characteristic nucleosome occupancy levels are strongly associated with the type and abundance of various AS events under normal and cold temperature conditions in Arabidopsis. Intriguingly, exitrons, alternatively spliced internal regions of protein-coding exons, exhibit distinctive nucleosome positioning pattern compared to other alternatively spliced regions. Likewise, nucleosome patterns differ between exitrons and retained introns, pointing to their distinct regulation. Collectively, our data show that characteristic changes in nucleosome positioning modulate AS in plants in response to cold.

Illuminating the dark side of the human transcriptome with long read transcript sequencing.

BACKGROUND: The human transcriptome annotation is regarded as one of the most complete of any eukaryotic species. However, limitations in sequencing technologies have biased the annotation toward multi-exonic protein coding genes. Accurate high-throughput long read transcript sequencing can now provide additional evidence for rare transcripts and genes such as mono-exonic and non-coding genes that were previously either undetectable or impossible to differentiate from sequencing noise. RESULTS: We developed the Transcriptome Annotation by Modular Algorithms (TAMA) software to leverage the power of long read transcript sequencing and address the issues with current data processing pipelines. TAMA achieved high sensitivity and precision for gene and transcript model predictions in both reference guided and unguided approaches in our benchmark tests using simulated Pacific Biosciences (PacBio) and Nanopore sequencing data and real PacBio datasets. By analyzing PacBio Sequel II Iso-Seq sequencing data of the Universal Human Reference RNA (UHRR) using TAMA and other commonly used tools, we found that the convention of using alignment identity to measure error correction performance does not reflect actual gain in accuracy of predicted transcript models. In addition, inter-read error correction can cause major changes to read mapping, resulting in potentially over 6 K erroneous gene model predictions in the Iso-Seq based human genome annotation. Using TAMA's genome assembly based error correction and gene feature evidence, we predicted 2566 putative novel non-coding genes and 1557 putative novel protein coding gene models. CONCLUSIONS: Long read transcript sequencing data has the power to identify novel genes within the highly annotated human genome. The use of parameter tuning and extensive output information of the TAMA software package allows for in depth exploration of eukaryotic transcriptomes. We have found long read data based evidence for thousands of unannotated genes within the human genome. More development in sequencing library preparation and data processing are required for differentiating sequencing noise from real genes in long read RNA sequencing data.

Downy Mildew effector HaRxL21 interacts with the transcriptional repressor TOPLESS to promote pathogen susceptibility.

Hyaloperonospora arabidopsidis (Hpa) is an oomycete pathogen causing Arabidopsis downy mildew. Effector proteins secreted from the pathogen into the plant play key roles in promoting infection by suppressing plant immunity and manipulating the host to the pathogen's advantage. One class of oomycete effectors share a conserved 'RxLR' motif critical for their translocation into the host cell. Here we characterize the interaction between an RxLR effector, HaRxL21 (RxL21), and the Arabidopsis transcriptional co-repressor Topless (TPL). We establish that RxL21 and TPL interact via an EAR motif at the C-terminus of the effector, mimicking the host plant mechanism for recruiting TPL to sites of transcriptional repression. We show that this motif, and hence interaction with TPL, is necessary for the virulence function of the effector. Furthermore, we provide evidence that RxL21 uses the interaction with TPL, and its close relative TPL-related 1, to repress plant immunity and enhance host susceptibility to both biotrophic and necrotrophic pathogens.

Nonsense-Mediated RNA Decay Factor UPF1 Is Critical for Posttranscriptional and Translational Gene Regulation in Arabidopsis.

Nonsense-mediated RNA decay (NMD) is an RNA control mechanism that has also been implicated in the broader regulation of gene expression. Nevertheless, a role for NMD in genome regulation has not yet been fully assessed, partially because NMD inactivation is lethal in many organisms. Here, we performed an in-depth comparative analysis of Arabidopsis (Arabidopsis thaliana) mutants lacking the NMD-related proteins UPF3, UPF1, and SMG7. We found different impacts of these proteins on NMD and the Arabidopsis transcriptome, with UPF1 having the biggest effect. Transcriptome assembly in UPF1-null plants revealed genome-wide changes in alternative splicing, suggesting that UPF1 functions in splicing. The inactivation of UPF1 led to translational repression, as manifested by a global shift in mRNAs from polysomes to monosomes and the downregulation of genes involved in translation and ribosome biogenesis. Despite these global changes, NMD targets and mRNAs expressed at low levels with short half-lives were enriched in the polysomes of upf1 mutants, indicating that UPF1/NMD suppresses the translation of aberrant RNAs. Particularly striking was an increase in the translation of TIR domain-containing, nucleotide binding, leucine-rich repeat (TNL) immune receptors. The regulation of TNLs via UPF1/NMD-mediated mRNA stability and translational derepression offers a dynamic mechanism for the rapid activation of TNLs in response to pathogen attack.

BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq.

BACKGROUND: The time required to analyse RNA-seq data varies considerably, due to discrete steps for computational assembly, quantification of gene expression and splicing analysis. Recent fast non-alignment tools such as Kallisto and Salmon overcome these problems, but these tools require a high quality, comprehensive reference transcripts dataset (RTD), which are rarely available in plants. RESULTS: A high-quality, non-redundant barley gene RTD and database (Barley Reference Transcripts - BaRTv1.0) has been generated. BaRTv1.0, was constructed from a range of tissues, cultivars and abiotic treatments and transcripts assembled and aligned to the barley cv. Morex reference genome (Mascher et al. Nature; 544: 427-433, 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al. Plant Physiol; 156: 20-28, 2011) determined transcript coverage, and high-resolution RT-PCR validated alternatively spliced (AS) transcripts of 86 genes in five different organs and tissue. These methods were used as benchmarks to select an optimal barley RTD. BaRTv1.0-Quantification of Alternatively Spliced Isoforms (QUASI) was also made to overcome inaccurate quantification due to variation in 5' and 3' UTR ends of transcripts. BaRTv1.0-QUASI was used for accurate transcript quantification of RNA-seq data of five barley organs/tissues. This analysis identified 20,972 significant differentially expressed genes, 2791 differentially alternatively spliced genes and 2768 transcripts with differential transcript usage. CONCLUSION: A high confidence barley reference transcript dataset consisting of 60,444 genes with 177,240 transcripts has been generated. Compared to current barley transcripts, BaRTv1.0 transcripts are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads. Precise transcript quantification using BaRTv1.0 allows routine analysis of gene expression and AS.

Application of a Sensitive and Reproducible Label-Free Proteomic Approach to Explore the Proteome of Individual Meiotic-Phase Barley Anthers.

Meiosis is a highly dynamic and precisely regulated process of cell division, leading to the production of haploid gametes from one diploid parental cell. In the crop plant barley (Hordeum vulgare), male meiosis occurs in anthers, in specialized cells called meiocytes. Barley meiotic tissue is scarce and not easily accessible, making meiosis study a challenging task. We describe here a new micro-proteomics workflow that allows sensitive and reproducible genome-wide label-free proteomic analysis of individual staged barley anthers. This micro-proteomic approach detects more than 4,000 proteins from such small amounts of material as two individual anthers, covering a dynamic range of protein relative abundance levels across five orders of magnitude. We applied our micro-proteomics workflow to investigate the proteome of the developing barley anther containing pollen mother cells in the early stages of meiosis and we successfully identified 57 known and putative meiosis-related proteins. Meiotic proteins identified in our study were found to be key players of many steps and processes in early prophase such as: chromosome condensation, synapsis, DNA double-strand breaks or crossover formation. Considering the small amount of starting material, this work demonstrates an important technological advance in plant proteomics and can be applied for proteomic examination of many size-limited plant specimens. Moreover, it is the first insight into the proteome of individual barley anther at early meiosis. The proteomic data have been deposited to the ProteomeXchange with the accession number PXD010887.