The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3\' processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for ANTHESIS PROMOTING FACTOR 1 (APRF1), a homolog of yeast Swd2 and human WDR82, known to regulate RNA polymerase II (RNA Pol II) during transcription termination. APRF1 interacts with TYPE ONE SERINE/THREONINE PROTEIN PHOSPHATASE 4 (TOPP4) (yeast Glc7/human PP1) and LUMINIDEPENDENS (LD), the latter showing structural features found in Ref2/PNUTS, all components of the yeast and human phosphatase module of the CPF 3\' end-processing machinery. LD has been shown to co-associate in vivo with the histone H3 K4 demethylase FLOWERING LOCUS D (FLD). This work shows how the APRF1/LD-mediated polyadenylation/termination process influences subsequent rounds of transcription by changing the local chromatin environment at FLC.

A well-established model for how plants start the process of flowering in periods of cold weather may need revisiting.

To synchronize flowering time with spring, many plants undergo vernalization, a floral-promotion process triggered by exposure to long-term winter cold. In Arabidopsis thaliana, this is achieved through cold-mediated epigenetic silencing of the floral repressor, FLOWERING LOCUS C (FLC). COOLAIR, a cold-induced antisense RNA transcribed from the FLC locus, has been proposed to facilitate FLC silencing. Here, we show that C-repeat (CRT)/dehydration-responsive elements (DREs) at the 3\'-end of FLC and CRT/DRE-binding factors (CBFs) are required for cold-mediated expression of COOLAIR. CBFs bind to CRT/DREs at the 3\'-end of FLC, both in vitro and in vivo, and CBF levels increase gradually during vernalization. Cold-induced COOLAIR expression is severely impaired in cbfs mutants in which all CBF genes are knocked-out. Conversely, CBF-overexpressing plants show increased COOLAIR levels even at warm temperatures. We show that COOLAIR is induced by CBFs during early stages of vernalization but COOLAIR levels decrease in later phases as FLC chromatin transitions to an inactive state to which CBFs can no longer bind. We also demonstrate that cbfs and FLCΔCOOLAIR mutants exhibit a normal vernalization response despite their inability to activate COOLAIR expression during cold, revealing that COOLAIR is not required for the vernalization process.
Long spells of cold winter weather may feel miserable, but they are often necessary for spring to blossom. Indeed, many plants need to face a prolonged period of low temperatures to be able to flower; this process is known as vernalization. While the molecular mechanisms which underpin vernalization are well-known, it is still unclear exactly how plants can ‘sense’ the difference between short and long periods of cold. Jeon, Jeong et al. set out to explore this question by focusing on COOLAIR, one of the rare genetic sequences identified as potentially being able to trigger vernalization. COOLAIR is a long noncoding RNA, a partial transcript of a gene that will not be ‘read’ by the cell to produce a protein but which instead regulates how and when certain genes are being switched on. COOLAIR emerges from the locus of the FLC gene, which is one of the main repressors of flowering, and it gradually accumulates in the plant when temperatures remain low for a long period. While some evidence suggests that COOLAIR may help to switch off FLC, other studies have raised some doubts about its involvement in vernalization. In response, Jeon, Jeong et al. examined the FLC gene in a range of plants closely related to A. thaliana, and in which COOLAIR also accumulates upon cold exposure. This helped them identify a class of proteins, known as CBFs, which could bind to sequences near the FLC gene to activate the production of COOLAIR when the plants were kept in cold conditions for a while. CBFs were already known to help plants adapt to short cold snaps, but these experiments confirmed that they could act as both short- and long-term cold sensors. This work allowed Jeon, Jeong et al. to propose a model in which CBF and therefore COOLAIR levels increase as the cold persists, until changes in the structure of the FLC gene prevent CBF from binding to it and COOLAIR production drops. Unexpectedly, examining the fate of mutants which could not produce COOLAIR revealed that these plants could still undergo vernalization, suggesting that the long noncoding RNA is in fact not necessary for this process. These results should prompt other scientists to further investigate the role of COOLAIR in vernalization; they also give insight into how coding and noncoding sequences may have evolved together in various members of the A. thaliana family to adapt to the environment.

FLOWERING LOCUS C (FLC), a MADS-box transcription factor, plays a major role in determining flowering time in Arabidopsis In this issue of Genes & Development, Zhao and colleagues (pp. 888-898) elucidate the role of COOLAIR antisense noncoding RNAs in FLC regulation through field trials and laboratory experiments. COOLAIR-mediated FLC silencing is induced by the first seasonal frost in the field and thus acts as a key molecular indicator during autumn for winter arrival.

Plants monitor many aspects of their fluctuating environments to help align their development with seasons. Molecular understanding of how noisy temperature cues are registered has emerged from dissection of vernalization in Arabidopsis, which involves a multiphase cold-dependent silencing of the floral repressor locus FLOWERING LOCUS C (FLC). Cold-induced transcriptional silencing precedes a low probability PRC2 epigenetic switching mechanism. The epigenetic switch requires the absence of warm temperatures as well as long-term cold exposure. However, the natural temperature inputs into the earlier transcriptional silencing phase are less well understood. Here, through investigation of Arabidopsis accessions in natural and climatically distinct field sites, we show that the first seasonal frost strongly induces expression of COOLAIR, the antisense transcripts at FLC Chamber experiments delivering a constant mean temperature with different fluctuations showed the freezing induction of COOLAIR correlates with stronger repression of FLC mRNA. Identification of a mutant that ectopically activates COOLAIR revealed how COOLAIR up-regulation can directly reduce FLC expression. Consistent with this, transgenes designed to knockout COOLAIR perturbed the early phase of FLC silencing. However, all transgenes designed to remove COOLAIR resulted in increased production of novel convergent FLC antisense transcripts. Our study reveals how natural temperature fluctuations promote COOLAIR regulation of FLC, with the first autumn frost acting as a key indicator of autumn/winter arrival.

Flowering is a critical stage of plant development and is closely correlated with seed production and crop yield. Flowering transition is regulated by complex genetic networks in response to endogenous and environmental signals. FLOWERING LOCUS C (FLC) is a central repressor in the flowering transition of Arabidopsis thaliana. The regulation of FLC expression is well studied at transcriptional and post-transcriptional levels. A subset of antisense transcripts from FLC locus, collectively termed cold-induced long antisense intragenic RNAs (COOLAIR), repress FLC expression under cold exposure. Recent studies have provided important insights into the alternative splicing of COOLAIR and FLC sense transcripts in response to developmental and environmental cues. Herein, at the 20th anniversary of FLC functional identification, we summarise new research advances in the alternative splicing of FLC sense and antisense transcripts that regulates flowering.

Long noncoding RNAs play important roles in plant epigenetic processes. While many extensive studies have delineated the range of their functions in plants, few detailed studies of the structure of plant long noncoding RNAs have been performed. Here, we review genome-wide and system-specific structural studies and describe methodology for structure determination.

Functional conservation of RNAs between different species is a key argument for their importance. While few long non-coding RNAs are conserved at the sequence level, many long non-coding RNAs have been identified that only share a position relative to other genes. It remains largely unknown whether and how these lncRNAs are conserved beyond their position. In Arabidopsis thaliana, the lncRNA COOLAIR is transcribed antisense from FLOWERING LOCUS C (FLC) in response to cold. Despite relatively low sequence similarity, the COOLAIR expression pattern and in vitro RNA secondary structure are highly conserved across the family Brassicaceae, which originated some 50 mya. It is unclear, however, whether COOLAIR functions in distantly related species such as monocots, which diverged some 150 mya. Here, we identified antisense lncRNAs from FLC homologs in various monocot species that share no sequence similarity with A. thaliana COOLAIR. Yet similar to COOLAIR, we found that BdODDSOC1 antisense (BdCOOLAIR1) and BdODDSOC2 antisense (BdCOOLAIR2) are induced by cold in a Brachypodium distachyon winter accession. Across B. distachyon accessions, the sequences of BdCOOLAIR1 and BdCOOLAIR2 are less conserved than exons but more conserved than flanking regions, suggesting a function for the transcript itself. Knock down of the BdODDSOC2 non-overlapping BdCOOLAIR2 transcript did not show a morphological phenotype, but did result in significantly higher BdODDSOC2 expression during cold, indicating that BdCOOLAIR2 performs a role in cis in the rate of BdODDSOC2 silencing. This functional similarity between eudicot and monocot species reveals ancient conservation or convergent evolution of FLC antisense transcription. Either scenario supports its functional importance.

Single-cell quantification of transcription kinetics and variability promotes a mechanistic understanding of gene regulation. Here, using single-molecule RNA fluorescence in situ hybridization and mathematical modeling, we dissect cellular RNA dynamics for Arabidopsis FLOWERING LOCUS C (FLC). FLC expression quantitatively determines flowering time and is regulated by antisense (COOLAIR) transcription. In cells without observable COOLAIR expression, we quantify FLC transcription initiation, elongation, intron processing, and lariat degradation, as well as mRNA release from the locus and degradation. In these heterogeneously sized cells, FLC mRNA number increases linearly with cell size, resulting in a large cell-to-cell variability in transcript level. This variation is accounted for by cell-size-dependent, Poissonian FLC mRNA production, but not by large transcriptional bursts. In COOLAIR-expressing cells, however, antisense transcription increases with cell size and contributes to FLC transcription decreasing with cell size. Our analysis therefore reveals an unexpected role for antisense transcription in modulating the scaling of transcription with cell size.

There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. We investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probing and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription.