Stochastic Variation in DNA Methylation Modulates Nucleosome Occupancy and Alternative Splicing in Arabidopsis thaliana.

Plants use complex gene regulatory mechanisms to overcome diverse environmental challenges. For instance, cold stress induces rapid and massive transcriptome changes via alternative splicing (AS) to confer cold tolerance in plants. In mammals, mounting evidence suggests chromatin structure can regulate co-transcriptional AS. Recent evidence also supports co-transcriptional regulation of AS in plants, but how dynamic changes in DNA methylation and the chromatin structure influence the AS process upon cold stress remains poorly understood. In this study, we used the DNA methylation inhibitor 5-Aza-2'-Deoxycytidine (5-aza-dC) to investigate the role of stochastic variations in DNA methylation and nucleosome occupancy in modulating cold-induced AS, in Arabidopsis thaliana (Arabidopsis). Our results demonstrate that 5-aza-dC derived stochastic hypomethylation modulates nucleosome occupancy and AS profiles of genes implicated in RNA metabolism, plant hormone signal transduction, and of cold-related genes in response to cold stress. We also demonstrate that cold-induced remodelling of DNA methylation regulates genes involved in amino acid metabolism. Collectively, we demonstrate that sudden changes in DNA methylation via drug treatment can influence nucleosome occupancy levels and modulate AS in a temperature-dependent manner to regulate plant metabolism and physiological stress adaptation.

Analysis of Predictors Affecting Biomechanical Function of the Knee Joint and Its Relation to Anterior Knee Pain.

BACKGROUND: Anterior knee pain is the most common problem in the young and sporting population. Quadriceps femoris angle and condylar distance are tools to assess the bio-mechanical function of the knee joint. The aim of this research was to give comparative data of quadriceps femoris angle and condylar distance in the Indian population (sedentary/sportsperson). The study also aims to know which parameter (condylar distance/quadriceps angle) is the better predictor for knee pain in the young Indian population. MATERIALS AND METHODS: This study was composed of a total of 130 individuals suffering from anterior knee pain which was divided into two categories; Sedentary and sportsperson. Each category consisted of 65 individuals. Q angle (goniometric method) and condylar distance (manual caliper) of each participant were calculated. A comparison of body parameters was done by independent t-test. Comparison between the two parameters (condylar distance and quadriceps angle) was done to know which is the better predictor of anterior knee pain. RESULTS: Statistically significant sexual variation (p<0.05) was observed in both quadriceps angle and condylar distance in sedentary and sportsperson groups. Females had a higher value of Q angle than males (p<0.05). The difference in quadriceps angle was statistically significant (p<0.05) between sedentary and sportsperson groups. Cohen's kappa coefficient of Q angle was 0.72 while that of bi-condylar distance was 0.49.  Conclusion: Q angle is a better indicator for anterior knee pain than condylar distance. Females in either category; sedentary and sportsperson, had higher Q angle in comparison to males making them more susceptible to disorders of the patellofemoral joint. Hence, encouragement and awareness are needed not only to carry out periodic screening of the susceptible population but also to emphasize its usage in clinical practice and the prognosis of the affected individual after treatment.

Epigenetic differences in an identical genetic background modulate alternative splicing in A. thaliana.

How stable and temperature-dependent variations in DNA methylation and nucleosome occupancy influence alternative splicing (AS) remains poorly understood in plants. To answer this, we generated transcriptome, whole-genome bisulfite, and MNase sequencing data for an epigenetic Recombinant Inbred Line (epiRIL) of A. thaliana at normal and cold temperature. For comparative analysis, the same data sets for the parental ecotype Columbia (Col-0) were also generated, whereas for DNA methylation, previously published high confidence methylation profiles of Col-0 were used. Significant epigenetic differences in an identical genetic background were observed between Col-0 and epiRIL lines under normal and cold temperatures. Our transcriptome data revealed that differential DNA methylation and nucleosome occupancy modulate expression levels of many genes and AS in response to cold. Collectively, DNA methylation and nucleosome levels exhibit characteristic patterns around intron-exon boundaries at normal and cold conditions, and any perturbation in them, in an identical genetic background is sufficient to modulate AS in Arabidopsis.

Rice Transcriptome Analysis Reveals Nitrogen Starvation Modulates Differential Alternative Splicing and Transcript Usage in Various Metabolism-Related Genes.

Nitrogen (N) is crucial for plant growth and development; however, excessive use of N fertilizers cause many problems including environmental damage, degradation of soil fertility, and high cost to the farmers. Therefore, immediate implementation is required to develop N efficient crop varieties. Rice being low nitrogen use efficiency (NUE) and a high demand staple food across the world has become a favorite crop to study the NUE trait. In the current study, we used the publicly available transcriptome data generated from the root and shoot tissues of two rice genotypes IR-64 and Nagina-22 (N-22) under optimum N supply (N+) and chronic N-starvation (N-). A stringent pipeline was applied to detect differentially expressed genes (DEGs), alternatively spliced (DAS) genes, differentially expressed transcripts (DETs) and differential transcript usage (DTU) transcripts in both the varieties and tissues under N+ and N- conditions. The DAS genes and DTU transcripts identified in the study were found to be involved in several metabolic and biosynthesis processes. We suggest alternative splicing (AS) plays an important role in fine-tuning the regulation of metabolic pathways related genes in genotype, tissue, and condition-dependent manner. The current study will help in understanding the transcriptional dynamics of NUE traits in the future.

MicroRNAs: Potential Targets for Developing Stress-Tolerant Crops.

Crop yield is challenged every year worldwide by changing climatic conditions. The forecasted climatic scenario urgently demands stress-tolerant crop varieties to feed the ever-increasing global population. Molecular breeding and genetic engineering approaches have been frequently exploited for developing crops with desired agronomic traits. Recently, microRNAs (miRNAs) have emerged as powerful molecules, which potentially serve as expression markers during stress conditions. The miRNAs are small non-coding endogenous RNAs, usually 20-24 nucleotides long, which mediate post-transcriptional gene silencing and fine-tune the regulation of many abiotic- and biotic-stress responsive genes in plants. The miRNAs usually function by specifically pairing with the target mRNAs, inducing their cleavage or repressing their translation. This review focuses on the exploration of the functional role of miRNAs in regulating plant responses to abiotic and biotic stresses. Moreover, a methodology is also discussed to mine stress-responsive miRNAs from the enormous amount of transcriptome data available in the public domain generated using next-generation sequencing (NGS). Considering the functional role of miRNAs in mediating stress responses, these molecules may be explored as novel targets for engineering stress-tolerant crop varieties.

A low noise cryogen-free scanning tunneling microscope-superconducting magnet system with vacuum sample transfer.

Imaging atomically resolved surfaces and performing spectroscopy of exotic surfaces at cryogenic temperature in the presence of the magnetic field is an engineering challenge. Additionally, performing these measurements in an all-cryogen-free environment compounds the above complexity due to the associated vibration and acoustic noise generated by the running of cryogenic cold heads. We here report successful integration of a cryogen-free scanning tunneling microscope (STM) with a cryogen-free superconducting vector-magnet, connected to an ultra-high vacuum cluster assembly for in situ sample transfer. We present details of the integration involving vibration and electrical noise isolation procedures allowing for operation of the STM at extremely low noise levels below 30 fA/Hz during normal operations of the complete vacuum-line assembly with multiple turbomolecular pumps. We demonstrate the above STM capability at cryogenic temperature and in the presence of the magnetic field through atomic resolution imaging of graphite and thin films of gold on the mica substrate transferred in situ to the STM chamber. We also demonstrate spectroscopy signatures of the superconducting gap in MgB2 thin films. The design of our in-house customized cluster-vacuum-line assembly provides unsought opportunities in continuous uninterrupted imaging of ultra-clean in-vacuum grown surfaces without the need for cryogenic refills in either the STM or the magnet.

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.

Genetic and Methylome Variation in Turkish Brachypodium Distachyon Accessions Differentiate Two Geographically Distinct Subpopulations.

Brachypodium distachyon (Brachypodium) is a non-domesticated model grass species that can be used to test if variation in genetic sequence or methylation are linked to environmental differences. To assess this, we collected seeds from 12 sites within five climatically distinct regions of Turkey. Seeds from each region were grown under standardized growth conditions in the UK to preserve methylated sequence variation. At six weeks following germination, leaves were sampled and assessed for genomic and DNA methylation variation. In a follow-up experiment, phenomic approaches were used to describe plant growth and drought responses. Genome sequencing and population structure analysis suggested three ancestral clusters across the Mediterranean, two of which were geographically separated in Turkey into coastal and central subpopulations. Phenotypic analyses showed that the coastal subpopulation tended to exhibit relatively delayed flowering and the central, increased drought tolerance as indicated by reduced yellowing. Genome-wide methylation analyses in GpC, CHG and CHH contexts also showed variation which aligned with the separation into coastal and central subpopulations. The climate niche modelling of both subpopulations showed a significant influence from the "Precipitation in the Driest Quarter" on the central subpopulation and "Temperature of the Coldest Month" on the coastal subpopulation. Our work demonstrates genetic diversity and variation in DNA methylation in Turkish accessions of Brachypodium that may be associated with climate variables and the molecular basis of which will feature in ongoing analyses.

Genome-Wide Identification of Splicing Quantitative Trait Loci (sQTLs) in Diverse Ecotypes of Arabidopsis thaliana.

Alternative splicing (AS) of pre-mRNAs contributes to transcriptome diversity and enables plants to generate different protein isoforms from a single gene and/or fine-tune gene expression during different development stages and environmental changes. Although AS is pervasive, the genetic basis for differential isoform usage in plants is still emerging. In this study, we performed genome-wide analysis in 666 geographically distributed diverse ecotypes of Arabidopsis thaliana to identify genomic regions [splicing quantitative trait loci (sQTLs)] that may regulate differential AS. These ecotypes belong to different microclimatic conditions and are part of the relict and non-relict populations. Although sQTLs were spread across the genome, we observed enrichment for trans-sQTL (trans-sQTLs hotspots) on chromosome one. Furthermore, we identified several sQTL (911) that co-localized with trait-linked single nucleotide polymorphisms (SNP) identified in the Arabidopsis genome-wide association studies (AraGWAS). Many sQTLs were enriched among circadian clock, flowering, and stress-responsive genes, suggesting a role for differential isoform usage in regulating these important processes in diverse ecotypes of Arabidopsis. In conclusion, the current study provides a deep insight into SNPs affecting isoform ratios/genes and facilitates a better mechanistic understanding of trait-associated SNPs in GWAS studies. To the best of our knowledge, this is the first report of sQTL analysis in a large set of Arabidopsis ecotypes and can be used as a reference to perform sQTL analysis in the Brassicaceae family. Since whole genome and transcriptome datasets are available for these diverse ecotypes, it could serve as a powerful resource for the biological interpretation of trait-associated loci, splice isoform ratios, and their phenotypic consequences to help produce more resilient and high yield crop varieties.

Alternative Splicing and Protein Diversity: Plants Versus Animals.

Plants, unlike animals, exhibit a very high degree of plasticity in their growth and development and employ diverse strategies to cope with the variations during diurnal cycles and stressful conditions. Plants and animals, despite their remarkable morphological and physiological differences, share many basic cellular processes and regulatory mechanisms. Alternative splicing (AS) is one such gene regulatory mechanism that modulates gene expression in multiple ways. It is now well established that AS is prevalent in all multicellular eukaryotes including plants and humans. Emerging evidence indicates that in plants, as in animals, transcription and splicing are coupled. Here, we reviewed recent evidence in support of co-transcriptional splicing in plants and highlighted similarities and differences between plants and humans. An unsettled question in the field of AS is the extent to which splice isoforms contribute to protein diversity. To take a critical look at this question, we presented a comprehensive summary of the current status of research in this area in both plants and humans, discussed limitations with the currently used approaches and suggested improvements to current methods and alternative approaches. We end with a discussion on the potential role of epigenetic modifications and chromatin state in splicing memory in plants primed with stresses.

Perspective on Alternative Splicing and Proteome Complexity in Plants.

Alternative splicing (AS) generates multiple transcripts from the same gene, however, AS contribution to proteome complexity remains elusive in plants. AS is prevalent under stress conditions in plants, but it is counterintuitive why plants would invest in protein synthesis under declining energy supply. We propose that plants employ AS not only to potentially increasing proteomic complexity, but also to buffer against the stress-responsive transcriptome to reduce the metabolic cost of translating all AS transcripts. To maximise efficiency under stress, plants may make fewer proteins with disordered domains via AS to diversify substrate specificity and maintain sufficient regulatory capacity. Furthermore, we suggest that chromatin state-dependent AS engenders short/long-term stress memory to mediate reproducible transcriptional response in the future.

Does co-transcriptional regulation of alternative splicing mediate plant stress responses?

Plants display exquisite control over gene expression to elicit appropriate responses under normal and stress conditions. Alternative splicing (AS) of pre-mRNAs, a process that generates two or more transcripts from multi-exon genes, adds another layer of regulation to fine-tune condition-specific gene expression in animals and plants. However, exactly how plants control splice isoform ratios and the timing of this regulation in response to environmental signals remains elusive. In mammals, recent evidence indicate that epigenetic and epitranscriptome changes, such as DNA methylation, chromatin modifications and RNA methylation, regulate RNA polymerase II processivity, co-transcriptional splicing, and stability and translation efficiency of splice isoforms. In plants, the role of epigenetic modifications in regulating transcription rate and mRNA abundance under stress is beginning to emerge. However, the mechanisms by which epigenetic and epitranscriptomic modifications regulate AS and translation efficiency require further research. Dynamic changes in the chromatin landscape in response to stress may provide a scaffold around which gene expression, AS and translation are orchestrated. Finally, we discuss CRISPR/Cas-based strategies for engineering chromatin architecture to manipulate AS patterns (or splice isoforms levels) to obtain insight into the epigenetic regulation of AS.

Transcriptome Analysis of Two Rice Varieties Contrasting for Nitrogen Use Efficiency under Chronic N Starvation Reveals Differences in Chloroplast and Starch Metabolism-Related Genes.

The nitrogen use efficiency (NUE) of crop plants is limited and enhancing it in rice, a major cereal crop, would be beneficial for farmers and the environment alike. Here we report the genome-wide transcriptome analysis of two rice genotypes, IR 64 (IR64) and Nagina 22 (N22) under optimal (+N) and chronic starvation (-N) of nitrogen (N) from 15-day-old root and shoot tissues. The two genotypes were found to be contrasting in their response to -N; IR64 root architecture and root dry weight remained almost equivalent to that under +N conditions, while N22 showed high foraging ability but a substantial reduction in biomass under -N. Similarly, the photosynthetic pigments showed a drastic reduction in N22 under low N, while IR64 was more resilient. Nitrate reductase showed significantly low specific activity under -N in both genotypes. Glutamate synthase (GOGAT) and citrate synthase CS activity were highly reduced in N22 but not in IR64. Transcriptome analysis of these genotypes revealed nearly double the number of genes to be differentially expressed (DEGs) in roots (1016) compared to shoots (571). The response of the two genotypes to N starvation was distinctly different reflecting their morphological/biochemical response with just two and eight common DEGs in the root and shoot tissues. There were a total of 385 nitrogen-responsive DEGs (106 in shoots and 279 in roots) between the two genotypes. Fifty-two of the 89 DEGs identified as specific to N22 root tissues were also found to be differentially expressed between the two genotypes under -N. Most of these DEGs belonged to starch and chloroplast metabolism, followed by membrane and signaling proteins. Physical mapping of DEGs revealed 95 DEGs in roots and 76 in shoots to be present in quantitative trait loci (QTL) known for NUE.

A Study of Endothelial Dysfunction in Patients of Non-Alcoholic Fatty Liver Disease.

INTRODUCTION: The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing worldwide rapidly and is regarded as the hepatic manifestation of metabolic syndrome. The present study was undertaken to study the endothelial dysfunction by flow mediated vasodilatation in NAFLD patients. MATERIAL & METHODS: 32 cases and 16 age and sex matched controls were included in the study. Flow mediated vasodilatation of the brachial artery was studied in both cases and controls. Anthropometric, clinical and biochemical assessment was also done. RESULTS: It was found that NAFLD patients had a significant endothelial dysfunction as assessed by flow mediated vasodilatation as compared with controls. Percentage change in FMD among NAFLD patients (13.54±3.65%) was found to be lower than that in controls (16.84±4.61%) and difference was found to be statistically significant (p 0.010). CONCLUSION: From the present study it can be concluded that NAFLD patients have significant endothelial dysfunction even in the absence of traditional risk factors of cardiovascular disease.

DeepSAGE based differential gene expression analysis under cold and freeze stress in seabuckthorn (Hippophae rhamnoides L.).

Seabuckthorn (Hippophae rhamnoides L.), an important plant species of Indian Himalayas, is well known for its immense medicinal and nutritional value. The plant has the ability to sustain growth in harsh environments of extreme temperatures, drought and salinity. We employed DeepSAGE, a tag based approach, to identify differentially expressed genes under cold and freeze stress in seabuckthorn. In total 36.2 million raw tags including 13.9 million distinct tags were generated using Illumina sequencing platform for three leaf tissue libraries including control (CON), cold stress (CS) and freeze stress (FS). After discarding low quality tags, 35.5 million clean tags including 7 million distinct clean tags were obtained. In all, 11922 differentially expressed genes (DEGs) including 6539 up regulated and 5383 down regulated genes were identified in three comparative setups i.e. CON vs CS, CON vs FS and CS vs FS. Gene ontology and KEGG pathway analysis were performed to assign gene ontology term to DEGs and ascertain their biological functions. DEGs were mapped back to our existing seabuckthorn transcriptome assembly comprising of 88,297 putative unigenes leading to the identification of 428 cold and freeze stress responsive genes. Expression of randomly selected 22 DEGs was validated using qRT-PCR that further supported our DeepSAGE results. The present study provided a comprehensive view of global gene expression profile of seabuckthorn under cold and freeze stresses. The DeepSAGE data could also serve as a valuable resource for further functional genomics studies aiming selection of candidate genes for development of abiotic stress tolerant transgenic plants.

Mining of microsatellites using next generation sequencing of seabuckthorn (Hippophae rhamnoides L.) transcriptome.

Gene based microsatellite markers are becoming more popular as compared to traditional random genomic microsatellite markers due to rapid and inexpensive method of isolation and their cross species portability. The present study documents occurrence of microsatellites in the transcriptome of seabuckthorn, a plant with immense medicinal, nutritional and ecological value. De novo assembly of over 80 million high quality short reads generated by high throughput next generation sequencing yielded 88297 putative unigenes. Of these, 7.69 % unigenes harbored microsatellite repeats with an average of one microsatellite per 6.704 Kb transcriptome. Dinucleotide repeats were most abundant followed by trinucleotide repeats. Microsatellites were densely populated in coding regions followed by 3' and 5' untranslated regions. AG and AAG type repeats were most frequently represented. Of the microsatellite positive unigenes, 48.81 % could be assigned gene ontology (GO) terms in order to assess associations between microsatellite containing unigenes and biological role of known genes. Utility of unigene specific microsatellites was assessed on the basis of polymorphism(s) detected in 18 seabuckthorn collections from Leh (India) using a set of randomly selected 25 unigene specific microsatellites. The findings presented here are likely to find immense use in future breeding and molecular biology research projects in seabuckthorn aiming at its overall development as a crop.

Optimization of de novo short read assembly of seabuckthorn (Hippophae rhamnoides L.) transcriptome.

Seabuckthorn (Hippophaerhamnoides L.) is known for its medicinal, nutritional and environmental importance since ancient times. However, very limited efforts have been made to characterize the genome and transcriptome of this wonder plant. Here, we report the use of next generation massive parallel sequencing technology (Illumina platform) and de novo assembly to gain a comprehensive view of the seabuckthorn transcriptome. We assembled 86,253,874 high quality short reads using six assembly tools. At our hand, assembly of non-redundant short reads following a two-step procedure was found to be the best considering various assembly quality parameters. Initially, ABySS tool was used following an additive k-mer approach. The assembled transcripts were subsequently subjected to TGICL suite. Finally, de novo short read assembly yielded 88,297 transcripts (> 100 bp), representing about 53 Mb of seabuckthorn transcriptome. The average length of transcripts was 610 bp, N50 length 1198 BP and 91% of the short reads uniquely mapped back to seabuckthorn transcriptome. A total of 41,340 (46.8%) transcripts showed significant similarity with sequences present in nr protein databases of NCBI (E-value < 1E-06). We also screened the assembled transcripts for the presence of transcription factors and simple sequence repeats. Our strategy involving the use of short read assembler (ABySS) followed by TGICL will be useful for the researchers working with a non-model organism's transcriptome in terms of saving time and reducing complexity in data management. The seabuckthorn transcriptome data generated here provide a valuable resource for gene discovery and development of functional molecular markers.