Cold-induced silencing by long antisense transcripts of an Arabidopsis Polycomb target.

Transcription in eukaryotic genomes generates an extensive array of non-protein-coding RNA, the functional significance of which is mostly unknown. We are investigating the link between non-coding RNA and chromatin regulation through analysis of FLC - a regulator of flowering time in Arabidopsis and a target of several chromatin pathways. Here we use an unbiased strategy to characterize non-coding transcripts of FLC and show that sense/antisense transcript levels correlate in a range of mutants and treatments, but change independently in cold-treated plants. Prolonged cold epigenetically silences FLC in a Polycomb-mediated process called vernalization. Our data indicate that upregulation of long non-coding antisense transcripts covering the entire FLC locus may be part of the cold-sensing mechanism. Induction of these antisense transcripts occurs earlier than, and is independent of, other vernalization markers and coincides with a reduction in sense transcription. We show that addition of the FLC antisense promoter sequences to a reporter gene is sufficient to confer cold-induced silencing of the reporter. Our data indicate that cold-induced FLC antisense transcripts have an early role in the epigenetic silencing of FLC, acting to silence FLC transcription transiently. Recruitment of the Polycomb machinery then confers the epigenetic memory. Antisense transcription events originating from 3' ends of genes might be a general mechanism to regulate the corresponding sense transcription in a condition/stage-dependent manner.

RNA 3' processing functions of Arabidopsis FCA and FPA limit intergenic transcription.

The RNA-binding proteins FCA and FPA were identified based on their repression of the flowering time regulator FLC but have since been shown to have widespread roles in the Arabidopsis thaliana genome. Here, we use whole-genome tiling arrays to show that a wide spectrum of genes and transposable elements are misexpressed in the fca-9 fpa-7 (fcafpa) double mutant at two stages of seedling development. There was a significant bias for misregulated genomic segments mapping to the 3' region of genes. In addition, the double mutant misexpressed a large number of previously unannotated genomic segments corresponding to intergenic regions. We characterized a subset of these misexpressed unannotated segments and established that they resulted from extensive transcriptional read-through, use of downstream polyadenylation sites, and alternative splicing. In some cases, the transcriptional read-through significantly reduced expression of the associated genes. FCA/FPA-dependent changes in DNA methylation were found at several loci, supporting previous associations of FCA/FPA function with chromatin modifications. Our data suggest that FCA and FPA play important roles in the A. thaliana genome in RNA 3' processing and transcription termination, thus limiting intergenic transcription.

Use of mRNA-seq to discriminate contributions to the transcriptome from the constituent genomes of the polyploid crop species Brassica napus.

BACKGROUND: Polyploidy often results in considerable changes in gene expression, both immediately and over evolutionary time. New phenotypes often arise with polyploid formation and may contribute to the fitness of polyploids in nature or their selection for use in agriculture. Oilseed rape (Brassica napus) is widely used to study the process of polyploidy both in artificially resynthesised and natural forms. mRNA-Seq, a recently developed approach to transcriptome profiling using deep-sequencing technologies is an alternative to microarrays for the study of gene expression in a polyploid. RESULTS: Illumina mRNA-Seq is comparable to microarray analysis for transcript quantification but has increased sensitivity and, very importantly, the potential to distinguish between homoeologous genes in polyploids. Using a novel curing process, we adapted a reference sequence that was a consensus derived from ESTs from both Brassica A and C genomes to one containing separate A and C genome versions for each of the 94,558 original unigenes. We aligned reads from B. napus to this cured reference, finding 38% more reads mapping from resynthesised lines and 28% more reads mapping from natural lines. Where the A and C versions differed at single nucleotide positions, termed inter-homoeologue polymorphisms (IHPs), we were able to apportion expression in the polyploid between the A and C genome homoeologues. 43,761 unigenes contained at least one IHP, with a mean frequency of 10.5 per kb unigene sequence. 6,350 of the unigenes with IHPs were differentially expressed between homoeologous gene pairs in resynthesised B. napus. 3,212 unigenes showed a similar pattern of differential expression across a range of natural B. napus crop varieties and, of these, 995 were in common with resynthesised B. napus. Functional classification showed over-representation in gene ontology categories not associated with dosage-sensitivity. CONCLUSION: mRNA-Seq is the method of choice for measuring transcript abundance in polyploids due to its ability to measure the contributions of homoeologues to gene expression. The identification of large numbers of differentially expressed genes in both a newly resynthesised polyploid and natural B. napus confirms that there are both immediate and long-term alterations in the expression of homoeologous gene pairs following polyploidy.

To what extent do callers follow the advice given by a non-emergency medical helpline (NHS 111): A retrospective cohort study.

National Health Service (NHS) 111 helpline was set up to improve access to urgent care in England, efficiency and cost-effectiveness of first-contact health services. Following trusted, authoritative advice is crucial for improved clinical outcomes. We examine patient and call-related characteristics associated with compliance with advice given in NHS 111 calls. The importance of health interactions that are not face-to-face has recently been highlighted by COVID-19 pandemic. In this retrospective cohort study, NHS 111 call records were linked to urgent and emergency care services data. We analysed data of 3,864,362 calls made between October 2013 and September 2017 relating to 1,964,726 callers across London. A multiple logistic regression was used to investigate associations between compliance with advice given and patient and call characteristics. Caller's action is 'compliant with advice given if first subsequent service interaction following contact with NHS 111 is consistent with advice given. We found that most calls were made by women (58%), adults aged 30-59 years (33%) and people in the white ethnic category (36%). The most common advice was for caller to contact their General Practitioner (GP) or other local services (18.2%) with varying times scales. Overall, callers followed advice given in 49% of calls. Compliance with triage advice was more likely in calls for children aged <16 years, women, those from Asian/Asian British ethnicity, and calls made out of hours. The highest compliance was among callers advised to self-care without the need to contact any other healthcare service. This is one of the largest studies to describe pathway adherence following telephone advice and associated clinical and demographic features. These results could inform attempts to improve caller compliance with advice given by NHS 111, and as the NHS moves to more hybrid way of working, the lessons from this study are key to the development of remote healthcare services going forward.

A newly-developed community microarray resource for transcriptome profiling in Brassica species enables the confirmation of Brassica-specific expressed sequences.

BACKGROUND: The Brassica species include an important group of crops and provide opportunities for studying the evolutionary consequences of polyploidy. They are related to Arabidopsis thaliana, for which the first complete plant genome sequence was obtained and their genomes show extensive, although imperfect, conserved synteny with that of A. thaliana. A large number of EST sequences, derived from a range of different Brassica species, are available in the public database, but no public microarray resource has so far been developed for these species. RESULTS: We assembled unigenes using approximately 800,000 EST sequences, mainly from three species: B. napus, B. rapa and B. oleracea. The assembly was conducted with the aim of co-assembling ESTs of orthologous genes (including homoeologous pairs of genes in B. napus from each of the A and C genomes), but resolving assemblies of paralogous, or paleo-homoeologous, genes (i.e. the genes related by the ancestral genome triplication observed in diploid Brassica species). 90,864 unique sequence assemblies were developed. These were incorporated into the BAC sequence annotation for the Brassica rapa Genome Sequencing Project, enabling the identification of cognate genomic sequences for a proportion of them. A 60-mer oligo microarray comprising 94,558 probes was developed using the unigene sequences. Gene expression was analysed in reciprocal resynthesised B. napus lines and the B. oleracea and B. rapa lines used to produce them. The analysis showed that significant expression could consistently be detected in leaf tissue for 35,386 unigenes. Expression was detected across all four genotypes for 27,355 unigenes, genome-specific expression patterns were observed for 7,851 unigenes and 180 unigenes displayed other classes of expression pattern. Principal component analysis (PCA) clearly resolved the individual microarray datasets for B. rapa, B. oleracea and resynthesised B. napus. Quantitative differences in expression were observed between the resynthesised B. napus lines for 98 unigenes, most of which could be classified into non-additive expression patterns, including 17 that showed cytoplasm-specific patterns. We further characterized the unigenes for which A genome-specific expression was observed and cognate genomic sequences could be identified. Ten of these unigenes were found to be Brassica-specific sequences, including two that originate from complex loci comprising gene clusters. CONCLUSION: We succeeded in developing a Brassica community microarray resource. Although expression can be measured for the majority of unigenes across species, there were numerous probes that reported in a genome-specific manner. We anticipate that some proportion of these will represent species-specific transcripts and the remainder will be the consequence of variation of sequences within the regions represented by the array probes. Our studies demonstrated that the datasets obtained from the arrays can be used for typical analyses, including PCA and the analysis of differential expression. We have also demonstrated that Brassica-specific transcripts identified in silico in the sequence assembly of public EST database accessions are indeed reported by the array. These would not be detectable using arrays designed using A. thaliana sequences.

Wheat blast: histopathology and transcriptome reprogramming in response to adapted and nonadapted Magnaporthe isolates.

* Blast disease (causal agent Magnaporthe oryzae) has presented as a new and serious field disease of wheat in South America. Here, we investigated the responses of wheat to both adapted and nonadapted isolates of the blast fungus Magnaporthe, examining cellular defence and transcriptional changes. * Resistance towards the nonadapted isolate was associated with the formation of appositions, here termed halos, beneath attempted Magnaporthe grisea penetration sites that wheat-adapted, M. oryzae isolates were able to breach. * Transcriptome analysis indicated extensive transcriptional reprogramming following inoculation with both wheat-adapted and nonadapted isolates of Magnaporthe. Functional annotation of many of the differentially expressed transcripts classified into the categories: cell rescue and defence, plant metabolism, cellular transport and regulation of transcription (although a significant number of transcripts remain unclassified). * Defence-related transcripts induced in common by adapted and nonadapted isolates were differentially regulated in response to M. oryzae and M. grisea isolates over time. Differential expression of genes involved in cellular transport indicated the importance of this process in plant defence. Functional characterisation of these transcripts and their role in defence may eventually lead to the identification of broad-spectrum resistance mechanisms in wheat towards Magnaporthe.

Complementary techniques of clustering and composite pattern analysis to Saccharomyces cerevisiae gene expression.

We present the combined application of clustering and composite pattern analysis to Saccharomyces cerevisiae gene expression and sequence data. The aim is to devise a system for the efficient investigation of the upstream regions associated with regulating the coordinated transcriptional response of genes via the location of cis-acting elements. The clustering algorithm presented allows clusters to overlap in light of the fact that many genes are regulated by multiple factors and/or are associated with multiple functions in the organism. The sequence clusters are analysed for shared monad and dyad patterns. These motifs are commonly regarded as putative cis-acting regulatory elements. Computationally, dyad patterns may be difficult and expensive to discover and verify if the sequences are very large. This has highlighted the need to obtain maximally--with regards to the pattern in question--'enriched' sets of sequences to uncover these motifs. We present overlapping clusters as an ideal basis for the discovery of conserved motifs.

Anthocyanins double the shelf life of tomatoes by delaying overripening and reducing susceptibility to gray mold.

Shelf life is an important quality trait for many fruit, including tomatoes. We report that enrichment of anthocyanin, a natural pigment, in tomatoes can significantly extend shelf life. Processes late in ripening are suppressed by anthocyanin accumulation, and susceptibility to Botrytis cinerea, one of the most important postharvest pathogens, is reduced in purple tomato fruit. We show that reduced susceptibility to B. cinerea is dependent specifically on the accumulation of anthocyanins, which alter the spreading of the ROS burst during infection. The increased antioxidant capacity of purple fruit likely slows the processes of overripening. Enhancing the levels of natural antioxidants in tomato provides a novel strategy for extending shelf life by genetic engineering or conventional breeding.

Expression profiling reveals COI1 to be a key regulator of genes involved in wound- and methyl jasmonate-induced secondary metabolism, defence, and hormone interactions.

The Arabidopsis gene COI1 is required for jasmonic acid (JA)-induced growth inhibition, resistance to insect herbivory, and resistance to pathogens. In addition, COI1 is also required for transcription of several genes induced by wounding or by JA. Here, we use microarray gene transcription profiling of wild type and coi1 mutant plants to examine the extent of the requirement of COI1 for JA-induced and wound-induced gene transcription. We show that COI1 is required for expression of approximately 84% of 212 genes induced by JA, and for expression of approximately 44% of 153 genes induced by wounding. Surprisingly, COI1 was also required for repression of 53% of 104 genes whose expression was suppressed by JA, and for repression of approximately 46% of 83 genes whose expression was suppressed by wounding. These results indicate that COI1 plays a pivotal role in wound- and JA signalling.