Background: Guanine-rich sequences are able to form complex RNA structures termed RNA G-quadruplexes in vitro. Because of their high stability, RNA Gquadruplexes are proposed to exist in vivo and are suggested to be associated with important biological relevance. However, there is a lack of direct evidence for RNA Gquadruplex formation in living eukaryotic cells. Therefore, it is unclear whether any purported functions are associated with the specific sequence content or the formation of an RNA G-quadruplex structure. Results: Using rG4-seq, we profile the landscape of those guanine-rich regions with the in vitro folding potential in the Arabidopsis transcriptome. We find a global enrichment of RNA G-quadruplexes with two G-quartets whereby the folding potential is strongly influenced by RNA secondary structures. Using in vitro and in vivo RNA chemical structure profiling, we determine that hundreds of RNA Gquadruplex structures are strongly folded in both Arabidopsis and rice, providing direct evidence of RNA G-quadruplex formation in living eukaryotic cells. Subsequent genetic and biochemical analyses show that RNA G-quadruplex folding is able to regulate translation and modulate plant growth. Conclusions: Our study reveals the existence of RNA G-quadruplex in vivo and indicates that RNA G-quadruplex structures act as important regulators of plant development and growth.

Background: Polyploidy is ubiquitous in eukaryotic plant and fungal lineages, and it leads to the coexistence of several copies of similar or related genomes in one nucleus. In plants, polyploidy is considered a major factor in successful domestication. However, polyploidy challenges chromosome folding architecture in the nucleus to establish functional structures. Results: We examine the hexaploid wheat nuclear architecture by integrating RNA-seq, ChIP-seq, ATAC-seq, Hi-C, and Hi-ChIP data. Our results highlight the presence of three levels of large-scale spatial organization: the arrangement into genome territories, the diametrical separation between facultative and constitutive heterochromatin, and the organization of RNA polymerase II around transcription factories. We demonstrate the micro-compartmentalization of transcriptionally active genes determined by physical interactions between genes with specific euchromatic histone modifications. Both intra-and interchromosomal RNA polymerase-associated contacts involve multiple genes displaying similar expression levels. Conclusions: Our results provide new insights into the physical chromosome organization of a polyploid genome, as well as on the relationship between epigenetic marks and chromosome conformation to determine a 3D spatial organization of gene expression, a key factor governing gene transcription in polyploids.

Antisense transcription through genic regions is pervasive in most genomes; however, its functional significance is still unclear. We are studying the role of antisense transcripts (COOLAIR) in the cold-induced, epigenetic silencing of Arabidopsis FLOWERING LOCUS C (FLC), a regulator of the transition to reproduction. Here we use single-molecule RNA FISH to address the mechanistic relationship of FLC and COOLAIR transcription at the cellular level. We demonstrate that while sense and antisense transcripts can co-occur in the same cell they are mutually exclusive at individual loci. Cold strongly upregulates COOLAIR transcription in an increased number of cells and through the mutually exclusive relationship facilitates shutdown of sense FLC transcription in cis. COOLAIR transcripts form dense clouds at each locus, acting to influence FLC transcription through changed H3K36me3 dynamics. These results may have general implications for other loci showing both sense and antisense transcription.

The requirement for vernalization, a need for prolonged cold to trigger flowering, aligns reproductive development with favorable spring conditions. In Arabidopsis thaliana vernalization depends on the cold-induced epigenetic silencing of the floral repressor locus FLC. Extensive natural variation in vernalization response is associated with A. thaliana accessions collected from different geographical regions. Here, we analyse natural variation for vernalization temperature requirement in accessions, including those from the northern limit of the A. thaliana range. Vernalization required temperatures above 0°C and was still relatively effective at 14°C in all the accessions. The different accessions had characteristic vernalization temperature profiles. One Northern Swedish accession showed maximum vernalization at 8°C, both at the level of flowering time and FLC chromatin silencing. Historical temperature records predicted all accessions would vernalize in autumn in N. Sweden, a prediction we validated in field transplantation experiments. The vernalization response of the different accessions was monitored over three intervals in the field and found to match that when the average field temperature was given as a constant condition. The vernalization temperature range of 0–14°C meant all accessions fully vernalized before snowfall in N. Sweden. These findings have important implications for understanding the molecular basis of adaptation and for predicting the consequences of climate change on flowering time.DOI: http://dx.doi.org/10.7554/eLife.06620.001

Liquid–liquid phase separation plays an important role in a variety of cellular processes, including the formation of membrane-less organelles, the cytoskeleton, signalling complexes, and many other biological supramolecular assemblies. Studies on the molecular basis of phase separation in cells have focused on protein-driven phase separation. In contrast, there is limited understanding on how RNA specifically contributes to phase separation. Here, we described a phase-separation-like phenomenon that SHORT ROOT (SHR) RNA undergoes in cells. We found that an RNA G-quadruplex (GQ) forms in SHR mRNA and is capable of triggering RNA phase separation under physiological conditions, suggesting that GQs might be responsible for the formation of the SHR phase-separation-like phenomenon in vivo. We also found the extent of GQ-triggered-phase-separation increases on exposure to conditions which promote GQ. Furthermore, GQs with more G-quartets and longer loops are more likely to form phase separation. Our studies provide the first evidence that RNA can adopt structural motifs to trigger and/or maintain the specificity of RNA-driven phase separation.

BackgroundDespite advances in other model organisms, there are currently no techniques to explore cell-to-cell variation and sub-cellular localization of RNA molecules at the single-cell level in plants.ResultsHere we describe a method for imaging individual mRNA molecules in Arabidopsis thaliana root cells using multiple singly labeled oligonucleotide probes. We demonstrate detection of both mRNA and nascent transcripts of the housekeeping gene Protein Phosphatase 2A. Our image analysis pipeline also enables quantification of mRNAs that reveals the frequency distribution of transcripts per cell underlying the population mean.ConclusionThis method allows single molecule RNA in situ to be exploited as a powerful tool for studying gene regulation in plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0114-x) contains supplementary material, which is available to authorized users.

SummaryMany organisms need to respond to complex, noisy environmental signals for developmental decision making. Here, we dissect how Arabidopsis plants integrate widely fluctuating field temperatures over month-long timescales to progressively upregulate VERNALIZATION INSENSITIVE3 (VIN3) and silence FLOWERING LOCUS C (FLC), aligning flowering with spring. We develop a mathematical model for vernalization that operates on multiple timescales—long term (month), short term (day), and current (hour)—and is constrained by experimental data. Our analysis demonstrates that temperature sensing is not localized to specific nodes within the FLC network. Instead, temperature sensing is broadly distributed, with each thermosensory process responding to specific features of the plants’ history of exposure to warm and cold. The model accurately predicts FLC silencing in new field data, allowing us to forecast FLC expression in changing climates. We suggest that distributed thermosensing may be a general property of thermoresponsive regulatory networks in complex natural environments.

In Arabidopsis thaliana, the cold-induced epigenetic regulation of FLOWERING LOCUS C (FLC) involves distinct phases of Polycomb repressive complex 2 (PRC2) silencing. During cold, a PHD-PRC2 complex metastably and digitally nucleates H3K27me3 within FLC. On return to warm, PHD-PRC2 spreads across the locus delivering H3K27me3 to maintain long-term silencing. Here, we studied natural variation in this process in Arabidopsis accessions, exploring Lov-1, which shows FLC reactivation on return to warm, a feature characteristic of FLC in perennial Brassicaceae. This analysis identifies an additional phase in this Polycomb silencing mechanism downstream from H3K27me3 spreading. In this long-term silencing (perpetuated) phase, the PHD proteins are lost from the nucleation region and silencing is likely maintained by the read-write feedbacks associated with H3K27me3. A combination of noncoding SNPs in the nucleation region mediates instability in this long-term silencing phase with the result that Lov-1 FLC frequently digitally reactivates in individual cells, with a probability that diminishes with increasing cold duration. We propose that this decrease in reactivation probability is due to reduced DNA replication after flowering. Overall, this work defines an additional phase in the Polycomb mechanism instrumental in natural variation of silencing, and provides avenues to dissect broader evolutionary changes at FLC.